Activation of bioluminescence by structural complementation

ABSTRACT

Provided herein are compositions and methods for the assembly of a bioluminescent complex from two or more non-luminescent (e.g., substantially non-luminescent) peptide and/or polypeptide units. In particular, bioluminescent activity is conferred upon a non-luminescent polypeptide via structural complementation with another, complementary non-luminescent peptide.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 16/023,972, filed Jun. 29, 2018, which is a continuation ofU.S. patent application Ser. No. 15/717,534, filed Sep. 27, 2017, whichis a continuation of U.S. patent application Ser. No. 15/073,249, filedMar. 17, 2016, now U.S. Pat. No. 9,869,670, which is a continuation ofU.S. patent application Ser. No. 14/209,610, filed Mar. 13, 2014, nowU.S. Pat. No. 9,797,890, which claims priority to U.S. ProvisionalPatent Application Ser. No. 61/791,549 filed Mar. 15, 2013, each ofwhich is hereby incorporated by reference in its entirety.

FIELD

Provided herein are compositions and methods for the assembly of abioluminescent complex from two or more non-luminescent (e.g.,substantially non-luminescent) peptide and/or polypeptide units. Inparticular, bioluminescent activity is conferred upon a non-luminescentpolypeptide via structural complementation with another, complementarynon-luminescent peptide.

BACKGROUND

Biological processes rely on covalent and non-covalent interactionsbetween molecules, macromolecules and molecular complexes. In order tounderstand such processes, and to develop techniques and compounds tomanipulate them for research, clinical and other practical applications,it is necessary to have tools available to detect and monitor theseinteractions. The study of these interactions, particularly underphysiological conditions (e.g. at normal expression levels formonitoring protein interactions), requires high sensitivity.

SUMMARY

The present invention relates to compositions comprising complementarynon-luminescent amino acid chains (e.g., substantially non-luminescentpeptides and/or polypeptides that are not fragments of a preexistingprotein), complexes thereof, and methods of generating an opticallydetectable bioluminescent signal upon association of the non-luminescentamino acid chains (e.g., peptides and/or polypeptides). In someembodiments, the present invention provides two or more non-luminescent,or substantially non-luminescent peptides and/or polypeptides, that,when brought together, assemble into a bioluminescent complex. In someembodiments, a pair of substantially non-luminescent peptide and/orpolypeptide units assembles into a bioluminescent complex. In otherembodiments, three or more substantially non-luminescent peptide and/orpolypeptide units assemble into a bioluminescent complex (e.g., ternarycomplex, tertiary complex, etc.). Provided herein are technologies fordetecting interactions between molecular entities (e.g., proteins,nucleic acids, carbohydrates, small molecules (e.g., small moleculelibraries)) by correlating such interactions to the formation of abioluminescent complex of otherwise non-luminescent (e.g., substantiallynon-luminescent) amino acid chains.

In some embodiments, the assembled pair catalyzes a chemical reaction ofan appropriate substrate into a high energy state, and light is emitted.In some embodiments, a bioluminescent complex exhibits luminescence inthe presence of substrate (e.g., coelenterazine, furimazine, etc.).

Although the embodiments described herein primarily describe and referto complementary, non-luminescent amino acid chains that formbioluminescent complexes, it is noted that the present technology canequally be applied to other detectable attributes (e.g., other enzymaticactivities, generation of a fluorophore, generation of a chromophore,etc.). The embodiments described herein relating to luminescence shouldbe viewed as applying to complementary, substantially non-enzymaticallyactive amino acid chains (e.g., peptides and/or polypeptides that arenot fragments of a preexisting protein) that separately lack a specifieddetectable activity (e.g., enzymatic activity) or substantiallynon-enzymatically active subunits of a polypeptide, complexes thereof,and methods of generating the detectable activity (e.g., an enzymaticactivity) upon association of the complementary, substantiallynon-enzymatically active amino acid chains (e.g., peptides and/orpolypeptides). Further, embodiments described herein that refer tonon-luminescent peptides and/or polypeptides are applied, in someembodiments, to substantially non-luminescent peptides and/orpolypeptides.

The invention is further directed to assays for the detection ofmolecular interactions between molecules of interest by linking theinteraction of a pair of non-luminescent peptides/polypeptides to theinteraction molecules of interest (e.g., transient association, stableassociation, complex formation, etc.). In such embodiments, a pair of anon-luminescent elements are tethered (e.g., fused) to molecules ofinterest and assembly of the bioluminescent complex is operated by themolecular interaction of the molecules of interest. If the molecules ofinterest engage in a sufficiently stable interaction, the bioluminescentcomplex forms, and a bioluminescent signal is generated. If themolecules of interest fail to engage in a sufficiently stableinteraction, the bioluminescent complex will not form or only formweakly, and a bioluminescent signal is not detectable or issubstantially reduced (e.g., substantially undetectable, essentially notdetectable, etc.). In some embodiments, the magnitude of the detectablebioluminescent signal is proportional (e.g., directly proportional) tothe amount, strength, favorability, and/or stability of the molecularinteractions between the molecules of interest.

In some embodiments, the present invention provides peptides comprisingan amino acid sequence having less than 100% (e.g., 20% . . . 30% . . .40% . . . 50% . . . 60% . . . 70% . . . 80%, or more) sequence identitywith SEQ ID NO: 2, wherein a detectable bioluminescent signal isproduced when the peptide contacts a polypeptide consisting of SEQ IDNO: 440. In some embodiments, the present invention provides peptidescomprising an amino acid sequence having less than 100% and greater than40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity with SEQ ID NO: 2, wherein a detectable bioluminescentsignal is produced when the peptide contacts a polypeptide consisting ofSEQ ID NO: 440. In some embodiments, a detectable bioluminescent signalis produced when the peptide contacts a polypeptide having less than100% and greater than 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity with SEQ ID NO: 440. In certain embodiments, thedetectable bioluminescent signal is produced, or is substantiallyincreased, when the peptide associates with the polypeptide comprisingor consisting of SEQ ID NO: 440, or a portion thereof. In preferredembodiments, the peptide exhibits alteration (e.g., enhancement) of oneor more traits compared to a peptide of SEQ ID NO: 2, wherein the traitsare selected from: affinity for the polypeptide consisting of SEQ ID NO:440, expression, intracellular solubility, intracellular stability andbioluminescent activity when combined with the polypeptide consisting ofSEQ ID NO: 440. Although not limited to these sequences, the peptideamino acid sequence may be selected from amino acid sequences of SEQ IDNOS: 3-438 and 2162-2365. In some embodiments, fusion polypeptides areprovided that comprise: (a) an above described peptide, and (b) a firstinteraction polypeptide that forms a complex with a second interactionpolypeptide upon contact of the first interaction polypeptide and thesecond interaction polypeptide. In certain embodiments, bioluminescentcomplexes are provided that comprise: (a) a first fusion polypeptidedescribed above and (b) a second fusion polypeptide comprising: (i) thesecond interaction polypeptide and (ii) a complement polypeptide thatemits a detectable bioluminescent signal when associated with thepeptide comprising an amino acid sequence having less than 100% andgreater than 40% sequence identity with SEQ ID NO: 2; wherein the firstfusion polypeptide and second fusion polypeptide are associated; andwherein the peptide comprising an amino acid sequence having less than100% and greater than 40% sequence identity with SEQ ID NO: 2 and thecomplement polypeptide are associated.

In some embodiments, the present invention provides polypeptidescomprising an amino acid sequence having less than 100% sequenceidentity with SEQ ID NO: 440, wherein a detectable bioluminescent signalis produced when the polypeptide contacts a peptide consisting of SEQ IDNO: 2. In some embodiments, the present invention provides polypeptidescomprising an amino acid sequence having less than 100% and greater than40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity with SEQ ID NO: 440, wherein a detectablebioluminescent signal is produced when the polypeptide contacts apeptide consisting of SEQ ID NO: 2. In some embodiments, a detectablebioluminescent signal is produced when the polypeptide contacts apeptide having less than 100% and greater than 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity with SEQ ID NO: 2. In some embodiments, thepolypeptide exhibits alteration (e.g., enhancement) of one or moretraits compared to a peptide of SEQ ID NO: 440, wherein the traits areselected from: affinity for the peptide consisting of SEQ ID NO: 2,expression, intracellular solubility, intracellular stability, andbioluminescent activity when combined with the peptide consisting of SEQID NO: 2. Although not limited to such sequences, the polypeptide aminoacid sequence may be selected from one of the amino acid sequences ofSEQ ID NOS: 441-2156. In some embodiments, the detectable bioluminescentsignal is produced when the polypeptide associates with the peptideconsisting of SEQ ID NO: 2. In some embodiments, a fusion polypeptide isprovided that comprises: (a) a polypeptide described above and (b) afirst interaction polypeptide that forms a complex with a secondinteraction polypeptide upon contact of the first interactionpolypeptide and the second interaction polypeptide. In certainembodiments, a bioluminescent complex is provided that comprises: (a) afirst fusion polypeptide described above; and (b) a second fusionpolypeptide comprising: (i) the second interaction polypeptide and (ii)a complement peptide that causes the polypeptide comprising an aminoacid sequence having less than 100% and greater than 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity with SEQ ID NO: 440 to emit a detectablebioluminescent signal when an association is formed between the two;wherein the first fusion polypeptide and second fusion polypeptide areassociated; and wherein the polypeptide comprising an amino acidsequence having less than 100% and greater than 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity with SEQ ID NO: 440 and the complement peptide areassociated.

In some embodiments, the present invention provides nucleic acids (e.g.,DNA, RNA, etc.), oligonucleotides, vectors, etc., that code for any ofthe peptides, polypeptides, fusion proteins, etc., described herein. Insome embodiments, a nucleic acid comprising or consisting of one of thenucleic acid sequences of SEQ ID NOS: 3-438 and 2162-2365 (coding fornon-luminescent peptides) and/or SEQ ID NOS 441-2156 (coding fornon-luminescent polypeptides) are provided. In some embodiments, othernucleic acid sequences coding for amino acid sequences of SEQ ID NOS:3-438 and 2162-2365 and/or SEQ ID NOS 441-2156 are provided.

In certain embodiments, the present invention provides bioluminescentcomplexes comprising: (a) a peptide comprising a peptide amino acidsequence having less than 100% sequence identity (e.g., >99%, <95%,<90%, <80%, <70%, <60%, <50%, etc.) with SEQ ID NO: 2; and (b) apolypeptide comprising a polypeptide amino acid sequence having lessthan 100% and greater than 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity with SEQ ID NO: 440, wherein the bioluminescentcomplex exhibits detectable luminescence. In certain embodiments, thepresent invention provides bioluminescent complexes comprising: (a) apeptide comprising a peptide amino acid sequence having less than 100%and greater than 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity with SEQ ID NO: 2; and (b) a polypeptide comprising apolypeptide amino acid sequence having less than 100% and greater than40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity with SEQ ID NO: 440, wherein the bioluminescentcomplex exhibits detectable luminescence. Although not limited toparticular sequences, in some embodiments, the peptide amino acidsequence is selected from one of the amino acid sequences provided inSEQ ID NOS: 3-438 and 2162-2365.

In various embodiments, bioluminescent complexes are provided thatcomprise: (a) a first amino acid sequence that is not a fragment of apreexisting protein; and (b) a second amino acid sequence that is not afragment of a preexisting protein, wherein the bioluminescent complexexhibits detectable luminescence, wherein the first amino acid sequenceand the second amino acid sequence are associated. Some suchbioluminescent complexes further comprise: (c) a third amino acidsequence comprising a first member of an interaction pair, wherein thethird amino acid sequence is covalently attached to the first amino acidsequence; and (d) a fourth amino acid sequence comprising a secondmember of an interaction pair, wherein the fourth amino acid sequence iscovalently attached to the second amino acid sequence. In certainembodiments, interactions (e.g., non-covalent interactions (e.g.,hydrogen bonds, ionic bonds, van der Waals forces, hydrophobicinteractions, etc.) covalent interactions (e.g., disulfide bonds), etc.)between the first amino acid sequence and the second amino acid sequencedo not significantly associate the first amino acid sequence and thesecond amino acid sequence in the absence of the interactions betweenthe first member and the second member of the interaction pair. In someembodiments, a first polypeptide chain comprises the first amino acidsequence and the third amino acid sequence, and wherein a secondpolypeptide chain comprises the second amino acid sequence and thefourth amino acid sequence. In some embodiments, the first polypeptidechain and the second polypeptide chain are expressed within a cell.

In some embodiments, the present invention provides a bioluminescentcomplex comprising: (a) a pair of non-luminescent elements, wherein eachnon-luminescent element is not a fragment of a preexisting protein; (b)an interaction pair, wherein each interaction element of the interactionpair is covalently attached to one of the non-luminescent elements.

Various embodiments described herein provide methods of detecting aninteraction between a first amino acid sequence and a second amino acidsequence comprising, for example, the steps of: (a) attaching the firstamino acid sequence to a third amino acid sequence and attaching thesecond amino acid sequence to a fourth amino acid sequence, wherein thethird and fourth amino acid sequences are not fragments of a preexistingprotein, wherein a complex of the third and fourth amino acid sequencesemits a detectable bioluminescent signal (e.g., substantially increasedbioluminescence relative to the polypeptide chains separately), whereinthe interactions (e.g., non-covalent) between the third and fourth aminoacid sequences are insufficient to form, or only weakly form, a complexof the third and fourth amino acid sequences in the absence ofadditional stabilizing and/or aggregating conditions, and wherein ainteraction between the first amino acid sequence and the second aminoacid sequence provides the additional stabilizing and/or aggregatingforces to produce a complex of the third and fourth amino acidsequences; (b) placing the first, second, third, and fourth amino acidsequences of step (a) in conditions to allow for interactions betweenthe first amino acid sequence and the second amino acid sequence tooccur; and (c) detecting the bioluminescent signal emitted by thecomplex of the third and fourth amino acid sequences, wherein detectionof the bioluminescent signal indicates an interaction between the firstamino acid sequence and the second amino acid sequence. In someembodiments, attaching the first amino acid sequence to the third aminoacid sequence and the second amino acid sequence to the fourth aminoacid sequence comprises forming a first fusion protein comprising thefirst amino acid sequence and the third amino acid sequence and forminga second fusion protein comprising the second amino acid sequence andthe fourth amino acid sequence. In some embodiments, the first fusionprotein and the second fusion protein further comprise linkers betweensaid first and third amino acid sequences and said second and fourthamino acid sequences, respectively. In certain embodiments, the firstfusion protein is expressed from a first nucleic acid sequence codingfor the first and third amino acid sequences, and the second fusionprotein is expressed from a second nucleic acid sequence coding for thesecond and fourth amino acid sequences. In some embodiments, a singlevector comprises the first nucleic acid sequence and the second nucleicacid sequence. In other embodiments, the first nucleic acid sequence andthe second nucleic acid sequence are on separate vectors. In certainembodiments, the steps of (a) “attaching” and (b) “placing” compriseexpressing the first and second fusion proteins within a cell.

Provided herein are methods of creating, producing, generating, and/oroptimizing a pair of non-luminescent elements comprising: (a) aligningthe sequences of three or more related proteins; (b) determining aconsensus sequence for the related proteins; (c) providing first andsecond fragments of a protein related to three or more proteins (orproviding first and second fragments of one of the three or moreproteins), wherein the fragments are individually substantiallynon-luminescent but exhibit luminescence upon interaction of thefragments; (d) mutating the first and second fragments at one or morepositions each, wherein the mutations alter the sequences of thefragments to be more similar to a corresponding portion of the consensussequence (e.g., wherein the mutating results in a pair ofnon-luminescent elements that are not fragments of a preexistingprotein), and (e) testing the pair of non-luminescent elements for theabsence (e.g., essential absence, substantial absence, etc.) ofluminescence when unassociated, and luminescence upon association of thenon-luminescent pair into a bioluminescent complex. Examples of such aprocess are described in Examples 1-5. In some embodiments, thenon-luminescent elements exhibit enhancement of one or more traitscompared to the first and second fragments, wherein the traits areselected from: increased reconstitution affinity, decreasedreconstitution affinity, enhanced expression, increased intracellularsolubility, increased intracellular stability, and increased intensityof reconstituted luminescence.

In some embodiments, the present invention provides detection reagentscomprising: (a) a polypeptide comprising an amino acid sequence havingless than 100% and greater than 40% sequence identity with SEQ ID NO:440, wherein a detectable bioluminescent signal is produced when thepolypeptide contacts a peptide consisting of SEQ ID NO: 2, and (b) asubstrate for a bioluminescent complex produced by the polypeptide and apeptide consisting of SEQ ID NO: 2. In some embodiments, the presentinvention provides detection reagents comprising: (a) a peptidecomprising an amino acid sequence having less than 100% sequenceidentity with SEQ ID NO: 2, wherein a detectable bioluminescent signalis produced when the peptide contacts a polypeptide consisting of SEQ IDNO: 440, and (b) a substrate for a bioluminescent complex produced bythe peptide and a polypeptide consisting of SEQ ID NO: 440. In someembodiments, the present invention provides detection reagentscomprising: (a) a peptide comprising an amino acid sequence having lessthan 100% and greater than 40% sequence identity with SEQ ID NO: 2,wherein a detectable bioluminescent signal is produced when the peptidecontacts a polypeptide consisting of SEQ ID NO: 440, and (b) a substratefor a bioluminescent complex produced by the peptide and a polypeptideconsisting of SEQ ID NO: 440.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph depicting the effect of various mutations of theGVTGWRLCKRISA (SEQ ID NO: 236) peptide on luminescence resulting fromcomplementation with SEQ ID NO: 440.

FIG. 2 shows a graph depicting the effect of various mutations of theSEQ ID NO: 440 polypeptide on luminescence resulting fromcomplementation with GVTGWRLCKRISA (SEQ ID NO: 236) or GVTGWRLFKRISA(SEQ ID NO: 108) peptides.

FIG. 3 (top) shows the luminescence (RLUs) detected in eachnon-luminescent polypeptide (NLpoly) mutant containing a single glycineto alanine substitution and (bottom) shows the fold increase inluminescence over wild-type.

FIG. 4 (top) show the luminescence (RLUs) detected in each NLpoly mutantcontaining a composite of glycine to alanine substitutions and (bottom)shows the fold increase in luminescence over wild-type.

FIG. 5 shows a graph depicting the luminescence (RLUs) detected inHT-NLpeptide fusions.

FIG. 6 shows a graph depicting the luminescence (RLUs) detected inHT-NLpep fusions.

FIG. 7 shows a graph depicting the luminescence (RLUs) detected inNLpeptide-HT fusions.

FIG. 8 shows the luminescence (RLUs) generated by a luminescent complexafter freeze-thaw cycles of non-luminescent peptide (NLpep).

FIG. 9 shows concentration normalized activity of peptides, and the TMRgel used to determine the relative concentrations.

FIG. 10 shows a graph of the luminescence of various mutations ofresidue R11 of NLpoly-5A2 in the presence of NLpep53 (top) and in theabsence of complimentary peptide (bottom).

FIG. 11 shows a graph of the luminescence of various mutations ofresidue A15 of NLpoly 5A2 in the presence of NLpep53 (top) and in theabsence of complimentary peptide (bottom).

FIG. 12 shows a graph of the luminescence of various mutations ofresidue L18 of NLpoly 5A2 in the presence of NLpep53 (top) and in theabsence of complimentary peptide (bottom).

FIG. 13 shows a graph of the luminescence of various mutations ofresidue F31 of NLpoly 5A2 in the presence of NLpep53 (top) and in theabsence of complimentary peptide (bottom).

FIG. 14 shows a graph of the luminescence of various mutations ofresidue V58 of NLpoly 5A2 in the presence of NLpep53 (top) and in theabsence of complimentary peptide (bottom).

FIG. 15 shows a graph of the luminescence of various mutations ofresidue A67 of NLpoly 5A2 in the presence of NLpep53 (top) and in theabsence of complimentary peptide (bottom).

FIG. 16 shows a graph of the luminescence of various mutations ofresidue M106 of NLpoly 5A2 in the presence of NLpep53 (top) and in theabsence of complimentary peptide (bottom).

FIG. 17 shows a graph of the luminescence of various mutations ofresidue L149 of NLpoly 5A2 in the presence of NLpep53 (top) and in theabsence of complimentary peptide (bottom).

FIG. 18 shows a graph of the luminescence of various mutations ofresidue V157 of NLpoly 5A2 in the presence of NLpep53 (top) and in theabsence of complimentary peptide (bottom).

FIG. 19 shows a graph of the luminescence of NLpep-HT fusions.

FIG. 20 shows a graph of the luminescence of NLpep-HT fusions, and a TMRgel indicating their relative expression levels.

FIG. 21 shows a graph of the luminescence of NLpep-HT fusions.

FIG. 22 shows a graph of the luminescence of NLpoly 5A2 (top) andNLpoly5A2+R11E in the presence of various NLpeps (bottom).

FIG. 23 shows a graph of the luminescence of NLpep-HT fusions.

FIG. 24 shows a graph of the luminescence of NLpolys 1-13 with NLpep53(top) and without complimentary peptide (bottom).

FIG. 25 shows a graph of the luminescence of various NLpolys withNLpep53 with NANOGLO or DMEM buffer and furimazine or coelenterazinesubstrate.

FIG. 26 shows a graph comparing luminescence in the presence of a ratioof furimazine with coelenterazine for various NLpolys and NLpep53.

FIG. 27 shows a graph comparing luminescence in the presence of a ratioof furimazine to coelenterazine for various NLpolys and NLpep53.

FIG. 28 shows a graph comparing luminescence in the presence offurimazine with coelenterazine for various NLpolys and NLpep53 in HEK293cell lysate.

FIG. 29 shows a graph of the luminescence of various combinations ofNLpoly and NLpep pairs in DMEM buffer with furimazine.

FIG. 30 shows a graph of the signal/background luminescence of variouscombinations of NLpoly and NLpep pairs in DMEM buffer with furimazine.

FIG. 31 shows a graph of luminescence and substrate specificity ofvarious NLpoly mutants with NLpep69 using either furimazine orcoelenterazine as a substrate.

FIG. 32 shows a comparison of luminescence and substrate specificity ofvarious NLpoly mutants with NLpep69 using either furimazine orcoelenterazine as a substrate, and under either lytic (bottom graph) orlive cell (top graph) conditions.

FIG. 33 shows a comparison of luminescence and substrate specificity ofNLpoly mutants with NLpep78 using either furimazine or coelenterazine asa substrate, and under either lytic (bottom graph) or live cell (topgraph) conditions.

FIG. 34 shows a comparison of luminescence and substrate specificity ofvarious NLpoly mutants with NLpep79 using either furimazine orcoelenterazine as a substrate, and under either lytic (bottom graph) orlive cell (top graph) conditions.

FIG. 35 shows graphs of the luminescence of NLpep78-HT (top) andNLpep79-HT (bottom) fusions in the presence of various NLpolys.

FIG. 36 shows a graph of the luminescence of various NLpolys in theabsence of NLpep.

FIG. 37 shows graphs of the luminescence of NLpep78-HT (top) andNLpep79-HT (bottom) fusions in the presence of various NLpolys witheither furimazine or coelenterazine substrates.

FIG. 38 shows a graph of the luminescence of NLpep78-HT with variousNLpolys expressed in CHO and HeLa cells.

FIG. 39 shows graphs of raw and normalized luminescence from NLpolyfused to firefly luciferase expressed in HEK293, Hela, and CHO celllysates.

FIG. 40 shows graphs of raw and normalized luminescence from NLpolyfused to click beetle red luciferase expressed in HEK293, Hela, and CHOcell lysates.

FIG. 41 shows a graphs of luminescence of complementation in live cellsusing either NLpoly wild-type or 5P.

FIG. 42 shows graphs of luminescence of cell-free complementation ofNLpep78-HT fusion (top) and NLpep79-HT fusion (bottom) with variousNLpolys.

FIG. 43 shows a graph of binding affinities for various combinations ofNLpeps and NLpolys expressed in HeLa, HEK293 and CHO cell lysate.

FIG. 44 shows a graph of binding affinities for various combinations ofNLpeps and NLpolys in PBS or NANOGLO buffer.

FIG. 45 shows a graph of binding affinities for NLpoly 5P with NLpep9(SEQ ID NO: 236) or NLpep53 (SEQ ID NO: 324) expressed in HeLa, HEK293or CHO cell lysate.

FIG. 46 shows a graph of luminescence of varying amounts of NLpolys inthe absence of NLpep.

FIG. 47 shows a graph of background luminescence of various NLpolyvariants.

FIG. 48 shows a graph of background luminescence of various NLpolyvariants.

FIG. 49 shows a SDS-PAGE gel of total lysate and soluble fraction ofseveral NLpoly variants

FIG. 50 shows (a) a SDS-PAGE gel of the total lysate and solublefraction of NLpoly variants and (b) background luminescence of NLpolyvariants.

FIG. 51 shows graphs of the luminescence generated with several NLpolyvariants when complemented with 10 nm (right) or 100 nM (left) ofNLpep78.

FIG. 52 shows graphs depicting background luminescence in E. coli lysateof various NLpoly variants.

FIG. 53 shows graphs depicting luminescence in E. coli lysate of variousNLpoly variants complemented with NLpep78.

FIG. 54 shows graphs depicting luminescence in E. coli lysate of variousNLpoly variants complemented with NLpep79.

FIG. 55 shows a graph of signal to background of various NLPolysvariants complemented with NLpep78 or NLpep79 and normalized to NLpoly5P.

FIG. 56 shows a graph depicting background, luminescence with NLpep79(right) or NLpep78 (left) and signal-to-noise or various NLpolyvariants.

FIG. 57 shows a SDS-PAGE gel of the total lysate and soluble fraction invarious NLpoly 5P variants.

FIG. 58 shows (A) the amount of total lysate and soluble fraction ofNLpoly 5P and NLpoly I107L, (B) luminescence generated by NLpoly 5P orNLpoly I107L without NLpep or with NLpep78 or NLpep79 and (C) theimproved signal-to-background of NLpoly I107L over NLpoly 5P.

FIG. 59 shows graphs of luminescence for various NLpoly variants (A)without complementary peptide, (B) with NLpep78-HT and (C) withNLpep79-HT.

FIG. 60 shows graphs of luminescence for various NLpoly variants (A)without complementary peptide, (B) with NLpep78-HT and (C) withNLpep79-HT.

FIG. 61 shows graphs of luminescence for various NLpoly variants (A)without complementary peptide, (B) with NLpep78-HT and (C) withNLpep79-HT.

FIG. 62 shows graphs of luminescence for various NLpoly variants (A)without complementary peptide, (B) with NLpep78-HT and (C) withNLpep79-HT.

FIG. 63 shows binding affinity between an elongated NLpoly variant(additional amino acids at the C-terminus) and a shortened NLpep(deleted amino acids at the N-terminus).

FIG. 64 shows a graph of binding affinity of various NLpoly variantswith NLpep78.

FIG. 65 shows the binding and Vmax of NLpep80 and NLpep87 to 5Pexpressed in mammalian cells (CHO, HEK293T and HeLa).

FIG. 66 shows the binding and Vmax of NLpep80 and NLpep87 to NLpoly 5Pexpressed in E. coli.

FIG. 67 shows a graph of luminescence of shortened NLpolys withelongated NLpeps.

FIG. 68 shows graphs of Kd and Vmax of NLpoly 5P in HeLa lysate withvarious complementary NLpeps.

FIG. 69 shows a graph of binding affinities for several NLpoly variantswith NLpep81.

FIG. 70 shows a graph of binding affinities for several NLpoly variantswith NLpep82.

FIG. 71 shows a graph of binding affinities for several NLpoly mutantswith NLpep78.

FIG. 72 shows a graph of Michaelis constants for several NLpoly mutantswith NLpep78.

FIG. 73 shows graphs of luminescence from a tertiary complementation oftwo NLpeps and NLpoly 5P-B9.

FIG. 74 shows a graph of luminescence of titration of NLpoly 5P withNLpep88-HT.

FIG. 75 shows images of intracellular localization of various NLpepfusions with HaloTag (HT).

FIG. 76 shows images of intracellular localization of NLpoly(wt) andNLpoly(5P).

FIG. 77 demonstrates the ability to detect via complementation anNLPep-conjugated protein of interest following separation by SDS-PAGEand transfer to a PVDF membrane.

FIG. 78 shows a graph of relative luminescent signal from various NLpolyvariants compared to NLpoly 5P (in the absence of NLpep).

FIG. 79 shows a graph of relative luminescent signal over backgroundfrom various NLpolys compared to NLpoly 5P (in the absence of NLpep).

FIG. 80 compares the dissociation constants for NLpeps consisting ofeither 1 (SEQ ID NO: 156) or 2 (SEQ ID NO: 2580) repeat units ofNLpep78.

FIG. 81 shows the affinity between NLpoly 5A2 and NLpep86.

FIG. 82 shows graphs of the luminescence from NLpoly variants withoutNLpep, with NLpep78, and NLpep79.

FIG. 83-90 show the dissociation constants as well as the Vmax valuesfor NLpoly 5A2, 5P, 8S and 11S with 96 variants of NLpeps. The uppergraphs show the values for SEQ ID Nos: 2366-2413. The lower graphs showthe values for SEQ ID NOs: 2414-2461.

FIG. 91 shows an image of a protein gel of total lysates and the solublefraction of the same lysate for NLpoly variants.

FIG. 92 shows an image of a protein gel of total lysates and the solublefraction of the same lysate for NLpoly variants as well as a tablecontaining the dissociation constants for the same variants.

FIG. 93 shows the substrate specificity for NLpoly 5P and 11S withNLpep79 and demonstrates that NLpoly11S has superior specificity forfurimazine than 5P.

FIG. 94 shows an image of a protein gel that follows the affinitypurification of NLpoly 8S through binding NLpep78.

FIG. 95 contains a table of the association and dissociation rateconstants for the binding of NLpoly WT or 11S to NLpepWT, 78 or 79.

FIG. 96 shows the Km values for various pairs of NLpoly/NLpep.

FIG. 97 compares the dissociation constant for NLpoly11S/NLpep79 atsub-saturating and saturating concentrations of furimazine.

FIG. 98 compares the Km values for NLpoly 5A2 with NLpepWT, 78 and 79.

FIG. 99 shows the luminescence of NLpolys from various steps in theevolution process in the absence of NLpep.

FIG. 100 shows the improvement in luminescence from E. coli-derivedNLpoly over the course of the evolution process with an overall ˜10⁵improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).

FIG. 101 shows the improvement in luminescence from HeLa-expressedNLpoly over the course of the evolution process with an overall ˜10⁵improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).

FIG. 102 shows the improvement in luminescence from HEK293cell-expressed NLpoly over the course of the evolution process with anoverall ˜10⁴ improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).

FIG. 103 shows dissociation constants and demonstrates a ˜10⁴ foldimprovement in binding affinity from NLpolyWT:NLpepWT toNLpoly11S:NLpep86.

FIG. 104 shows an image of a protein gel of total lysates and thesoluble fraction of the same lysate for NLpoly variants from varioussteps of the evolution process.

FIG. 105 shows luminescence of various NLpolys in the absence of NLpepand in the presence of NLpep78 and NLpep79.

FIG. 106 shows luminescence of various NLpolys in the absence of NLpepand in the presence of NLpep78 and NLpep79.

FIG. 107 shows luminescence of various NLpolys in the absence of NLpepand in the presence of NLpep78 and NLpep79.

FIG. 108 shows a comparison of luminescence generated by cellsexpressing different combinations of FRB and FKBP fused to NLpoly5P andNLpep80/87 after 15 min treatment with rapamycin or vehicle. Foldinduction refers to signal generated in the presence of rapamycincompared to signal generated with vehicle.

FIG. 109 shows a comparison of luminescence generated by cellsexpressing different combinations of FRB and FKBP fused to NLpoly5P andNLpep80/87 after 60 min treatment with rapamycin or vehicle.

FIG. 110 shows a comparison of luminescence generated by cellsexpressing different combinations of FRB and FKBP fused to NLpoly5P andNLpep80/87 after 120 min treatment with rapamycin or vehicle.

FIG. 111 shows a comparison of luminescence generated by cellsexpressing different combinations of FRB and FKBP fused to NLpoly5P andNLpep80/87 after 120 min treatment with rapamycin or vehicle. All 8possible combinations of FRB and FKBP fused to NLpoly/NLpep were testedand less total DNA was used.

FIG. 112 shows a comparison of luminescence generated by FRB or FKBPfusions expressed in the absence of binding partner.

FIG. 113 shows a comparison of luminescence generated by cellstransfected with varying amounts of FRB-NLpoly5P and FKBP-NLpep80/87DNA.

FIG. 114 shows a comparison of luminescence generated by cellstransfected with varying amounts of FRB-NLpoly5P or FKBP-NLpep80/87 DNAin the absence of binding partner.

FIG. 115 shows a comparison of luminescence generated by cellstransfected with varying amounts of FRB-NLpoly5P and FKBP-NLpep80/87DNA. This example differs from FIG. 113 in that lower levels of DNA wereused.

FIG. 116 shows a comparison of luminescence generated by cellstransfected with varying amounts of FRB-NLpoly5P or FKBP-NLpep80/87 DNAin the absence of binding partner. This differs from FIG. 114 in thatlower levels of DNA were used.

FIG. 117 shows a comparison of luminescence generated by cellstransfected with varying amounts of FRB-NLpoly5P and FKBP-NLpep80 DNAafter treatment with rapamycin for different lengths of time.

FIG. 118 shows a comparison of luminescence generated by cellstransfected with varying amounts of FRB-NLpoly5P and FKBP-NLpep87 DNAafter treatment with rapamycin for different lengths of time.

FIG. 119 shows a comparison of luminescence generated by cellsexpressing different combinations of FRB-NLpoly5P withFKBP-NLpep80/87/95/96/97. Assay was performed in both a two-day andthree-day format.

FIG. 120 shows a comparison of luminescence generated by cellsexpressing different combinations of FRB-NLpoly5A2 withFKBP-NLpep80/87/95/96/97. Assay was performed in both a two-day andthree-day format.

FIG. 121 shows a comparison of luminescence generated by cellsexpressing different combinations of FRB-NLpoly5A2 or FRB-NLpoly 11 Swith FKBP-NLpep101/104/105/106/107/108/109/110.

FIG. 122 shows a comparison of luminescence generated by cellstransfected with different combinations of FRB-NLpoly5A2 or FRB-NLpoly11 S with FKBP-NLpep87/96/98/99/100/101/102/103.

FIG. 123 shows a comparison of luminescence generated by cellstransfected with different levels of FRB-NLpoly11S andFKBP-NLpep87/101/102/107 DNA.

FIG. 124 shows a comparison of luminescence generated by cellstransfected with different levels of FRB-NLpoly5A2 andFKBP-NLpep87/101/102/107 DNA.

FIG. 125 shows a rapamycin dose response curve showing luminescence ofcells expressing FRB-NLpoly5P and FKBP-NLpep80/87 DNA.

FIG. 126 shows a rapamycin dose response curve showing luminescence ofcells expressing FRB-NLpoly5A2 or FRB-NLpoly 11S and FKBP-NLpep87/101DNA.

FIG. 127 shows a comparison of luminescence generated by cellsexpressing FRB-11S and FKBP-101 and treated with substrate PBI-4377 orfurimazine.

FIG. 128 shows a rapamycin time course of cells expressingFRB-NLpoly11S/5A2 and FKBP-NLpep87/101 conducted in the presence orabsence of rapamycin wherein the rapamycin was added manually.

FIG. 129 shows a rapamycin time course of cells expressingFRB-NLpoly11S/5A2 and FKBP-NLpep87/101 conducted in the presence orabsence of rapamycin wherein the rapamycin was added via instrumentinjector.

FIG. 130 shows luminescence generated by FRB-NLpoly11S and FKBP-NLpep101as measured on two different luminescence-reading instruments.

FIG. 131 provides images showing luminescence of cells expressingFRB-NLpoly11S and FKBP-NLpep101 at various times after treatment withrapamycin.

FIG. 132 provides a graph showing Image J quantitation of the signalgenerated by individual cells expressing FRB-NLpoly11S and FKBP-NLpep101at various times after treatment with rapamycin.

FIG. 133 shows a comparison of luminescence in different cell linesexpressing FRB-NLpoly11S and FKBP-NLpep101.

FIG. 134 shows a comparison of luminescence generated by cellsexpressing FRB-NLpoly11S and FKBP-NLpep101 after treatment with therapamycin competitive inhibitor FK506.

FIG. 135 shows (left side) luminescence generated by cells expressingFRB-NLpoly11S and FKBP-NLpep101 after treatment with the rapamycincompetitive inhibitor FK506, and (right side) the percent ofluminescence remaining after treatment with FK506.

FIG. 136 shows luminescence generated by cells transfected withdifferent combinations of V2R-NLpoly5A2 or V2R-NLpoly11S withNLpep87/101-ARRB2 in the presence or absence of the V2R agonist AVP.

FIG. 137 shows an AVP treatment time course showing luminescencegenerated by cells transfected with V2R-NLpoly11S and NLpep87/101-ARRB2after treatment with AVP wherein AVP was added manually.

FIG. 138 shows an AVP treatment time course showing luminescencegenerated by cells transfected with different combinations ofV2R-NLpoly5A2 or V2R-NLpoly11S with NLpep87/101-ARRB2 after treatmentwith AVP wherein AVP was added via instrument injector.

FIG. 139 shows an AVP treatment time course at 37° C. showingluminescence generated by cells expressing different configurations ofV2R and ARRB2 fused to NLpoly 11 S and NLpep101 after treatment withAVP.

FIG. 140 shows a comparison of luminescence in different cell linesexpressing V2R-NLpep 11S and NLpep101-ARRB2.

FIG. 141 shows 60× images showing luminescence of cells expressingV2R-NLpoly11S and NLpep101-ARRB2 at various times after treatment withAVP.

FIG. 142 shows 150× images showing luminescence of cells expressingV2R-NLpoly11S and NLpep101-ARRB2 at various times after treatment withAVP.

FIG. 143 shows a protein gel of total lysates and the soluble fractionof the same lysate for NLpoly variants.

FIG. 144 shows the dissociation constants for NLpoly 5P and combinationsof mutations at positions 31, 46, 75, 76, and 93 in NLpoly 5P.

FIG. 145 shows a transferase example of post translational modificationenzyme activity detection using an NLpep and aminopeptidase.

FIG. 146 shows a hydrolase example of post translational modificationenzyme activity detection using an NLpep and methyl-specific antibody.

FIG. 147 contains wavelength scans for NLpoly WT complemented witheither NLpepWT or NLpepWT conjugated to TMR.

FIG. 148 contains wavelength scans for NanoLuc fused to HaloTag (NL-HT)and NLpoly 5A2 complemented with NLPepWT with 4 additional amino acids(DEVD) and conjugated to Non-chloroTOM (NCT) (SEQ ID NO: 2579).

FIG. 149 shows a schematic a tertiary interaction wherein the energytransfer with an NLpoly and NLpep can also be used to measure threemolecules interacting. In the schematic, a GPCR labeled with an NLpolyand a GPCR interacting protein labeled with an NLpep form abioluminescent complex when they interact. This allows measurement ofthe binary interaction. If a small molecule GPCR ligand bearing anappropriate fluorescent moiety for energy transfer interacts with thissystem, energy transfer will occur. Therefore, the binaryprotein-protein interaction and the ternary drug-protein-proteininteraction can be measured in the same experiment.

FIG. 150 shows a graph and table of binding affinities of NLpoly11S tosynthetic NLPep78 and NLPep78 at the N- or C-terminus of a fusionpartner (HaloTag).

FIG. 151 shows a graph and table of binding affinities of NLpoly11S tosynthetic NLPep79 and NLPep79 at the N- or C-terminus of a fusionpartner (HaloTag).

FIG. 152 shows a graph depicting normalized fluorescence intensity ofNLpoly11S with NLPep86 or PBI-4877.

FIG. 153 shows a graph depicting normalized fluorescence intensity ofNLpoly11S with NLPep86 or PBI-5434.

FIG. 154 shows a graph depicting normalized fluorescence intensity ofNLpoly11S with NLPep86 or PBI-5436.

FIG. 155 shows a graph demonstrating furimazine binding affinity inaffinity buffer of complexes between NLpoly11S and NLpep86, 78, 99, 101,104, 128 and 114.

FIG. 156 shows a graph demonstrating furimazine binding affinity inNanoGlo assay buffer of complexes between NLpoly11S and NLpep86, 78, 99,101, 104, 128 and 114.

FIG. 157 shows graphs depicting the change in affinity (NLpoly156/NLPep1and NLpoly11S/NLPep1) with increasing concentrations of furimazinesubstrate.

FIG. 158 shows graphs depicting the change in affinity (NLpoly156/NLPep1and NLpoly11S/NLPep1) with increasing concentrations of NLPep1.

FIG. 159 shows a graph depicting Vmax and Bmax NLPoly156, NLPoly11S, andNanoLuc® luciferase (Nluc) with NLPep1.

FIG. 160 shows a graph depicting RLU as a function of NLPepconcentration for NLPoly156 and NLPep86, 78, 79, 99, 101, 104, 114, 128and wt.

FIG. 161 shows a Western blot depicting expression level in HEK293Tcells of NLPoly156 and NLPoly11S compared to full-length NanoLuc®luciferase.

FIG. 162 shows graphs depicting a comparison of the affinity of theβ-lactamase SME and its inhibitor BLIPY50A as unfused proteins or whenfused to NLPoly11S and NLPep114.

FIG. 163 shows a comparison of luminescence generated by cellsexpressing different combinations of FRB-NLpoly11S withFKBP-NLpep101/111-136

FIG. 164 shows a comparison of luminescence generated by cellsexpressing different combinations of FRB-NLpoly11S with FKBP-NLpep114and 137-143.

FIG. 165 shows rapamycin dose response curves of cells expressingFRB-NLpoly11S and FKBP-NLpep78/79/99/101/104/114/128

FIG. 166 shows response of cells expressing FRB-NLpoly11S andFKBP-78/79/99/101/104/114/128 to the rapamycin competitive inhibitorFK506

FIG. 167 shows a comparison of luminescence generated by cellstransfected with different ratios of FRB-NLpoly11S and FKBP-NLpep114.

FIG. 168 shows a comparison of luminescence generated by cellsexpressing NLpoly11S/NLpep114 fusions of FRB/FKBP in differentorientations and with different linker lengths.

FIG. 169 shows graphs depicting rapamycin (A) dose-specific and (B)time-specific induction of FRB-NLpoly11S/FKBP-NLpep114 or split fireflycomplementation signals.

FIG. 170 shows graphs depicting FK506(A) dose-specific and (B)time-specific inhibition of FRB-NLpoly11S/FKBP-NLpep114 or split fireflycomplementation signals.

FIG. 171 shows Western blots depicting similar expression levels ofFKBP-NLpep114 and FKBP-Fluc(394-544) at equal levels of transfected DNA.

FIG. 172 shows graphs depicting (A) dose-specific and (B) time-specificinhibition of NLpoly11S-BRD4 and Histone H3.3-NLpep114 interaction byIBET-151.

FIG. 173 shows a graph depicting dose dependent increases in RAS/CRAF,BRAF/BRAF and CRAF/BRAF dimerization in response to BRAF inhibitorGDC0879.

FIG. 174 shows a graph depicting RLU as a function of NLPepconcentration for NLpoly11S and NLpep86 (SEQ ID NO: 390), wt (SEQ ID NO:2578), and NLpep114 (SEQ ID NO: 2271).

FIG. 175 shows a schematic of an assay utilizing a high affinity peptideof a luminescent pair as an intracellular protein tag and thepolypeptide of the luminescent pair as a detection reagent.

FIG. 176 shows a graph demonstrating the linear range of the affinity ofNLpoly11S and MLpep86.

FIG. 177 shows images demonstrating the sensitivity of detectingproteins tagged with a high affinity NLPep using 11S. This figure alsocompares the detection using NLPep/NLPoly to the detection usingfluorescently labeled HaloTag.

FIG. 178 shows a graph demonstrating the stability of NLpoly11S.

FIG. 179 shows a graph demonstrating the linear range of the affinity ofNLpoly11S and NLpep78.

FIG. 180 shows a summary of NLpep sequences. High affinity (spontaneous)peptides are those peptides (NLpep) which bind to NLpoly11S with highaffinity. Dark/Quencher peptides are those peptides (NLpep) which canreduce the levels of light being produced or detected from NLpoly11S.

FIG. 181 shows a schematic for the concept of structural complementationwhere the LSP and SSP (i.e., NLpoly and NLpep) are brought together toproduce a bioluminescent signal (panels A, B). Upon disruption of aprotein interaction (i.e. X and Y), LSP and SSP come apart resulting ina decrease in luminescence (Panel C).

FIGS. 182A-B show two options (A, B) for engineering structuralcomplementation to be a loss of signal upon protein interaction betweenX and Y and a gain of signal upon disruption of the interaction betweenX and Y. Option A represents intermolecular structural complementation.Option B represents intramolecular structural complementation. FIG. 182Bshows a list of genetic constructs that could be suitable forintramolecular structural complementation.

FIG. 183 shows (A) inhibition of NLpoly11S and NLpep114 binding byvarious dark peptides, and (B) dose-dependent inhibition by Lys-162 andGln-162 peptides.

FIG. 184 A shows that inhibition by Q-162 and A-162 is dose-dependent.Panel B shows that Q-162 produces a signal on its own in adose-dependent manner, while the dose-dependency of A-162 is subtle atbest.

FIG. 185 shows graphs demonstrating dose-response of the dark peptideswith CP Nluc.

FIG. 186 shows graphs depicting a time course of dark peptide with CPNluc.

FIG. 187 shows the dark peptide dose-dependent inhibition ofluminescence generated from FRB-NLpoly11S alone and also betweenFRB-NLpoly11S and FKBP-NLpep114 in the presence and absence ofrapamycin.

FIG. 188 shows the dark peptide dose-dependent inhibition ofluminescence generated from either FRB-NanoLuc (311) or NanoLuc-FRB(307) in the presence and absence of rapamycin (RLU).

FIG. 189 shows the dark peptide dose-dependent inhibition ofluminescence generated from either FRB-NanoLuc (311) or NanoLuc-FRB(307) in the presence and absence of rapamycin (normalized to no darkpeptide control; 100%).

FIG. 190 shows that the dark peptides, when fused to FKBP, can competewith both low (114) and high (80) affinity peptides (also FKBP fusions)and as a result reduce the total luminescence being produced anddetected in live cells.

FIG. 191 shows the signal comparison between Fluc and NLpep86-basedassays for intracellular levels of Fluc. The table depicts SEQ ID Nos:172, 390, and 2581-2585.

FIG. 192 shows graphs demonstrating the utility of tandem linked NLpepsin complementing Npoly11S.

FIG. 193 shows a graph demonstrating that NLpoly and NLpep components donot interfere with intracellular degradation of reporter protein FlucP.

FIG. 194 shows a schematic demonstrating and extracellular proteaseactivity assay.

FIG. 195 shows a schematic of an assay for measuring the activity of anenzyme using a ProNLpep.

FIG. 196 shows a schematic of an assay for screening antibodies,proteins, peptides or transporters that mediate cellularinternalization.

FIG. 197 shows a schematic of a post-translational modificationtransferase assay.

FIG. 198 shows a schematic of a post-translational modificationhydrolase assay.

FIG. 199 shows graphs correlating Tyrosine Kinase SRC activity withluminescence over background in a post-translational modification assay.

FIG. 200 shows a graph depicting spontaneous complementation of threedifferent versions of NLpoly11S with twelve synthetic peptides.

FIG. 201 shows a schematic of a homogeneous immunoassay format utilizingfusions of NLpep and NLpoly with separate binding moieties A and B.

FIG. 202 shows graphs demonstrating: (A) reduction in backgroundluminescence from NLpoly11S upon complex formation with GWALFKK (SEQ IDNO: 2351) and Dabcyl-GWALFKK (SEQ ID NO: 2351), and (B) NLpep86 forms acomplex with NLpoly11S in the presence of GWALFKK (SEQ ID NO: 2351) andDabcyl-GWALFKK (SEQ ID NO: 2351).

FIG. 203 shows graphs demonstrating: (A) VTGWALFEEIL (SEQ ID NO: 2372)(Trp 11mer) and VTGYALFEEIL (SEQ ID NO: 2355) (Tyr 11mer) induceluminescence over background (NLpoly11S alone; no peptide control), andthat the N-terminal Dabcyl versions of each provide significantquenching of this signal, and (B) that NLpep86 forms a complex withNLpoly11S in the presence of Dabcyl versions of Trp 11mer and Tyr 11mer.

DEFINITIONS

As used herein, the term “substantially” means that the recitedcharacteristic, parameter, and/or value need not be achieved exactly,but that deviations or variations, including for example, tolerances,measurement error, measurement accuracy limitations and other factorsknown to skill in the art, may occur in amounts that do not preclude theeffect the characteristic was intended to provide. A characteristic orfeature that is substantially absent (e.g., substantiallynon-luminescent) may be one that is within the noise, beneathbackground, below the detection capabilities of the assay being used, ora small fraction (e.g., <1%, <0.1%, <0.01%, <0.001%, <0.00001%,<0.000001%, <0.0000001%) of the significant characteristic (e.g.,luminescent intensity of a bioluminescent protein or bioluminescentcomplex).

As used herein, the term “bioluminescence” refers to production andemission of light by a chemical reaction catalyzed by, or enabled by, anenzyme, protein, protein complex, or other biomolecule (e.g.,bioluminescent complex). In typical embodiments, a substrate for abioluminescent entity (e.g., bioluminescent protein or bioluminescentcomplex) is converted into an unstable form by the bioluminescententity; the substrate subsequently emits light.

As used herein the term “complementary” refers to the characteristic oftwo or more structural elements (e.g., peptide, polypeptide, nucleicacid, small molecule, etc.) of being able to hybridize, dimerize, orotherwise form a complex with each other. For example, a “complementarypeptide and polypeptide” are capable of coming together to form acomplex. Complementary elements may require assistance to form a complex(e.g., from interaction elements), for example, to place the elements inthe proper conformation for complementarity, to co-localizecomplementary elements, to lower interaction energy for complementary,etc.

As used herein, the term “complex” refers to an assemblage or aggregateof molecules (e.g., peptides, polypeptides, etc.) in direct and/orindirect contact with one another. In one aspect, “contact,” or moreparticularly, “direct contact” means two or more molecules are closeenough so that attractive noncovalent interactions, such as Van der Waalforces, hydrogen bonding, ionic and hydrophobic interactions, and thelike, dominate the interaction of the molecules. In such an aspect, acomplex of molecules (e.g., a peptide and polypeptide) is formed underassay conditions such that the complex is thermodynamically favored(e.g., compared to a non-aggregated, or non-complexed, state of itscomponent molecules). As used herein the term “complex,” unlessdescribed as otherwise, refers to the assemblage of two or moremolecules (e.g., peptides, polypeptides or a combination thereof).

As used herein, the term “non-luminescent” refers to an entity (e.g.,peptide, polypeptide, complex, protein, etc.) that exhibits thecharacteristic of not emitting a detectable amount of light in thevisible spectrum (e.g., in the presence of a substrate). For example, anentity may be referred to as non-luminescent if it does not exhibitdetectable luminescence in a given assay. As used herein, the term“non-luminescent” is synonymous with the term “substantiallynon-luminescent. For example, a non-luminescent polypeptide (NLpoly) issubstantially non-luminescent, exhibiting, for example, a 10-fold ormore (e.g., 100-fold, 200-fold, 500-fold, 1×10³-fold, 1×10⁴-fold,1×10⁵-fold, 1×10⁶-fold, 1×10⁷-fold, etc.) reduction in luminescencecompared to a complex of the NLpoly with its non-luminescent complementpeptide. In some embodiments, an entity is “non-luminescent” if anylight emission is sufficiently minimal so as not to create interferingbackground for a particular assay.

As used herein, the terms “non-luminescent peptide” (e.g., NLpep) and“non-luminescent polypeptide” (e.g., NLpoly) refer to peptides andpolypeptides that exhibit substantially no luminescence (e.g., in thepresence of a substrate), or an amount that is beneath the noise, or a10-fold or more (e.g., 100-fold, 200-fold, 500-fold, 1×10³-fold,1×10⁴-fold, 1×10⁵-fold, 1×10⁶-fold, 1×10⁷-fold, etc.) when compared to asignificant signal (e.g., luminescent complex) under standard conditions(e.g., physiological conditions, assay conditions, etc.) and withtypical instrumentation (e.g., luminometer, etc.). In some embodiments,such non-luminescent peptides and polypeptides assemble, according tothe criteria described herein, to form a bioluminescent complex. As usedherein, a “non-luminescent element” is a non-luminescent peptide ornon-luminescent polypeptide. The term “bioluminescent complex” refers tothe assembled complex of two or more non-luminescent peptides and/ornon-luminescent polypeptides. The bioluminescent complex catalyzes orenables the conversion of a substrate for the bioluminescent complexinto an unstable form; the substrate subsequently emits light. Whenuncomplexed, two non-luminescent elements that form a bioluminescentcomplex may be referred to as a “non-luminescent pair.” If abioluminescent complex is formed by three or more non-luminescentpeptides and/or non-luminescent polypeptides, the uncomplexedconstituents of the bioluminescent complex may be referred to as a“non-luminescent group.”

As used herein, the term “interaction element” refers to a moiety thatassists in bringing together a pair of non-luminescent elements or anon-luminescent group to form a bioluminescent complex. In a typicalembodiment, a pair of interaction elements (a.k.a. “interaction pair”)is attached to a pair of non-luminescent elements (e.g., non-luminescentpeptide/polypeptide pair), and the attractive interaction between thetwo interaction elements facilitates formation of the bioluminescentcomplex; although the present invention is not limited to such amechanism, and an understanding of the mechanism is not required topractice the invention. Interaction elements may facilitate formation ofthe bioluminescent complex by any suitable mechanism (e.g., bringingnon-luminescent pair/group into close proximity, placing anon-luminescent pair/group in proper conformation for stableinteraction, reducing activation energy for complex formation,combinations thereof, etc.). An interaction element may be a protein,polypeptide, peptide, small molecule, cofactor, nucleic acid, lipid,carbohydrate, antibody, etc. An interaction pair may be made of two ofthe same interaction elements (i.e. homopair) or two differentinteraction elements (i.e. heteropair). In the case of a heteropair, theinteraction elements may be the same type of moiety (e.g., polypeptides)or may be two different types of moieties (e.g., polypeptide and smallmolecule). In some embodiments, in which complex formation by theinteraction pair is studied, an interaction pair may be referred to as a“target pair” or a “pair of interest,” and the individual interactionelements are referred to as “target elements” (e.g., “target peptide,”“target polypeptide,” etc.) or “elements of interest” (e.g., “peptide ofinterest,” “polypeptide or interest,” etc.).

As used herein, the term “preexisting protein” refers to an amino acidsequence that was in physical existence prior to a certain event ordate. A “peptide that is not a fragment of a preexisting protein” is ashort amino acid chain that is not a fragment or sub-sequence of aprotein (e.g., synthetic or naturally-occurring) that was in physicalexistence prior to the design and/or synthesis of the peptide.

As used herein, the term “fragment” refers to a peptide or polypeptidethat results from dissection or “fragmentation” of a larger whole entity(e.g., protein, polypeptide, enzyme, etc.), or a peptide or polypeptideprepared to have the same sequence as such. Therefore, a fragment is asubsequence of the whole entity (e.g., protein, polypeptide, enzyme,etc.) from which it is made and/or designed. A peptide or polypeptidethat is not a subsequence of a preexisting whole protein is not afragment (e.g., not a fragment of a preexisting protein). A peptide orpolypeptide that is “not a fragment of a preexisting bioluminescentprotein” is an amino acid chain that is not a subsequence of a protein(e.g., natural or synthetic) that: (1) was in physical existence priorto design and/or synthesis of the peptide or polypeptide, and (2)exhibits substantial bioluminescent activity.

As used herein, the term “subsequence” refers to peptide or polypeptidethat has 100% sequence identify with another, larger peptide orpolypeptide. The subsequence is a perfect sequence match for a portionof the larger amino acid chain.

As used herein, the term “sequence identity” refers to the degree twopolymer sequences (e.g., peptide, polypeptide, nucleic acid, etc.) havethe same sequential composition of monomer subunits. The term “sequencesimilarity” refers to the degree with which two polymer sequences (e.g.,peptide, polypeptide, nucleic acid, etc.) have similar polymersequences. For example, similar amino acids are those that share thesame biophysical characteristics and can be grouped into the families,e.g., acidic (e.g., aspartate, glutamate), basic (e.g., lysine,arginine, histidine), non-polar (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan) anduncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). The “percent sequence identity” (or “percentsequence similarity”) is calculated by: (1) comparing two optimallyaligned sequences over a window of comparison (e.g., the length of thelonger sequence, the length of the shorter sequence, a specifiedwindow), (2) determining the number of positions containing identical(or similar) monomers (e.g., same amino acids occurs in both sequences,similar amino acid occurs in both sequences) to yield the number ofmatched positions, (3) dividing the number of matched positions by thetotal number of positions in the comparison window (e.g., the length ofthe longer sequence, the length of the shorter sequence, a specifiedwindow), and (4) multiplying the result by 100 to yield the percentsequence identity or percent sequence similarity. For example, ifpeptides A and B are both 20 amino acids in length and have identicalamino acids at all but 1 position, then peptide A and peptide B have 95%sequence identity. If the amino acids at the non-identical positionshared the same biophysical characteristics (e.g., both were acidic),then peptide A and peptide B would have 100% sequence similarity. Asanother example, if peptide C is 20 amino acids in length and peptide Dis 15 amino acids in length, and 14 out of 15 amino acids in peptide Dare identical to those of a portion of peptide C, then peptides C and Dhave 70% sequence identity, but peptide D has 93.3% sequence identity toan optimal comparison window of peptide C. For the purpose ofcalculating “percent sequence identity” (or “percent sequencesimilarity”) herein, any gaps in aligned sequences are treated asmismatches at that position.

As used herein, the term “physiological conditions” encompasses anyconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, chemical makeup, etc. thatare compatible with living cells.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Sample may also refer tocell lysates or purified forms of the peptides and/or polypeptidesdescribed herein. Cell lysates may include cells that have been lysedwith a lysing agent or lysates such as rabbit reticulocyte or wheat germlysates. Sample may also include cell-free expression systems.Environmental samples include environmental material such as surfacematter, soil, water, crystals and industrial samples. Such examples arenot however to be construed as limiting the sample types applicable tothe present invention.

As used herein, unless otherwise specified, the terms “peptide” and“polypeptide” refer to polymer compounds of two or more amino acidsjoined through the main chain by peptide amide bonds (—C(O)NH—). Theterm “peptide” typically refers to short amino acid polymers (e.g.,chains having fewer than 25 amino acids), whereas the term “polypeptide”typically refers to longer amino acid polymers (e.g., chains having morethan 25 amino acids).

DETAILED DESCRIPTION

The study of protein interactions, particularly under physiologicalconditions and/or at physiologic expression levels, requires highsensitivity. In particular embodiments described herein, proteininteractions with small molecules, nucleic acids, other proteins, etc.are detected based on the association of two non-luminescent elementsthat come together to from a bioluminescent complex capable of producinga detectable signal (e.g., luminescence). The formation of thebioluminescent complex is dependent upon the protein interaction that isbeing monitored.

Provided herein are compositions and methods for the assembly of abioluminescent complex from two or more non-luminescent peptide and/orpolypeptide units (e.g., non-luminescent pair). In some embodiments, thenon-luminescent peptide and/or polypeptide units are not fragments of apreexisting protein (e.g., are not complementary subsequences of a knownpolypeptide sequence). In particular, bioluminescent activity isconferred upon a non-luminescent polypeptide via structuralcomplementation with a non-luminescent peptide.

In some embodiments, provided herein are non-luminescent pairs for usein detecting and monitoring molecular interactions (e.g.,protein-protein, protein-DNA, protein-RNA interactions, RNA-DNA,protein-small molecule, RNA-small-molecule, etc.). Also provided hereinare complementary panels of interchangeable non-luminescent elements(e.g., peptides and polypeptides) that have variable affinities andluminescence upon formation of the various bioluminescent complexes(e.g., a high-affinity/high-luminescence pair, amoderate-affinity/high-luminescence pair, alow-affinity/moderate-luminescence pair, etc.). Utilizing differentcombinations of non-luminescent elements provides an adaptable systemcomprising various pairs ranging from lower to higher affinities,luminescence and other variable characteristics. This adaptabilityallows the detection/monitoring of molecular interactions to befine-tuned to the specific molecule(s) of interest and expands the rangeof molecular interactions that can be monitored to include interactionswith very high or low affinities. Further provided herein are methods bywhich non-luminescent pairs (or groups) and panels of non-luminescentpairs (or groups) are developed and tested.

In some embodiments, the interaction between the peptide/polypeptidemembers of the non-luminescent pair alone is insufficient to form thebioluminescent complex and produce the resulting bioluminescent signal.However, if an interaction element is attached to eachpeptide/polypeptide member of the non-luminescent pair, then theinteractions of the interaction pair (e.g., to form an interactioncomplex) facilitate formation of the bioluminescent complex. In suchembodiments, the bioluminescent signal from the bioluminescent complex(or the capacity to produce such a signal in the presence of substrate)serves as a reporter for the formation of the interaction complex. If aninteraction complex is formed, then a bioluminescent complex is formed,and a bioluminescent signal is detected/measured/monitored (e.g., in thepresence of substrate). If an interaction complex fails to form (e.g.,due to unfavorable conditions, due to unstable interaction between theinteraction elements, due to incompatible interaction elements), then abioluminescent complex does not form, and a bioluminescent signal is notproduced.

In certain embodiments, the interaction pair comprises two molecules ofinterest (e.g., proteins of interest). For example, assays can beperformed to detect the interaction of two molecules of interest bytethering each one to a separate member of a non-luminescent pair. Ifthe molecules of interest interact (e.g., transiently interact, stablyinteract, etc.), the non-luminescent pair is brought into closeproximity in a suitable conformation and a bioluminescent complex isformed (and bioluminescent signal is produced/detected (in the presenceof substrate)). In the absence of an interaction between the moleculesof interest (e.g., no complex formation, not even transient interaction,etc.), the non-luminescent pair does not interact in a sufficientmanner, and a bioluminescent signal is not produced or only weaklyproduced. Such embodiments can be used to study the effect of inhibitorson complex formation, the effect of mutations on complex formation, theeffect of conditions (e.g., temperature, pH, etc.) on complex formation,the interaction of a small molecule (e.g., potential therapeutic) with atarget molecule, etc.

Different non-luminescent pairs may require different strength, durationand/or stability of the interaction complex to result in bioluminescentcomplex formation. In some embodiments, a stable interaction complex isrequired to produce a detectable bioluminescent signal. In otherembodiments, even a weak or transient interaction complex results inbioluminescent complex formation. In some embodiments, the strength orextent of an interaction complex is directly proportional to thestrength of the resulting bioluminescent signal. Some non-luminescentpairs produce a detectable signal when combined with an interactioncomplex with a high millimolar dissociation constant (e.g., K_(d)>100mM). Other non-luminescent pairs require an interaction pair with a lowmillimolar (e.g., K_(d)<100 mM), micromolar (e.g., K_(d)<1 mM),nanomolar (e.g., K_(d)<1 μM), or even picomolar (e.g., K_(d)<1 nM)dissociation constant in order to produce a bioluminescent complex witha detectable signal.

In some embodiments, one or more of the non-luminescentpeptides/polypeptides are not fragments of a pre-existing protein. Insome embodiments, one or more of the non-luminescentpeptides/polypeptides are not fragments of a pre-existing bioluminescentprotein. In some embodiments, neither/none of the non-luminescentpeptides/polypeptides are fragments of a pre-existing protein. In someembodiments, neither/none of the non-luminescent peptides/polypeptidesare fragments of a pre-existing bioluminescent protein. In someembodiments, neither the non-luminescent peptide nor non-luminescentpolypeptide that assemble together to form a bioluminescent complex arefragments of a pre-existing protein. In some embodiments, anon-luminescent element for use in embodiments of the present inventionis not a subsequence of a preexisting protein. In some embodiments, anon-luminescent pair for use in embodiments described herein does notcomprise complementary subsequences of a preexisting protein.

In some embodiments, non-luminescent peptides/polypeptides aresubstantially non-luminescent in isolation. In certain embodiments, whenplaced in suitable conditions (e.g., physiological conditions),non-luminescent peptides/polypeptides interact to form a bioluminescentcomplex and produce a bioluminescent signal in the presence ofsubstrate. In other embodiments, without the addition of one or moreinteraction elements (e.g., complementary interaction elements attachedto the component non-luminescent peptide and non-luminescentpolypeptide), non-luminescent peptides/polypeptides are unable to form abioluminescent complex or only weakly form a complex. In suchembodiments, non-luminescent peptides/polypeptides are substantiallynon-luminescent in each other's presence alone, but produce significantdetectable luminescence when aggregated, associated, oriented, orotherwise brought together by interaction elements. In some embodiments,without the addition of one or more interaction elements (e.g.,complementary interaction elements attached to the component peptide andpolypeptide), peptides and/or polypeptides that assemble into thebioluminescent complex produce a low level of luminescence in eachother's presence, but undergo a significant increase in detectableluminescence when aggregated, associated, oriented, or otherwise broughttogether by interaction elements.

In some embodiments, compositions and methods described herein compriseone or more interaction elements. In a typical embodiment, aninteraction element is a moiety (e.g., peptide, polypeptide, protein,small molecule, nucleic acid, lipid, carbohydrate, etc.) that isattached to a peptide and/or polypeptide to assemble into thebioluminescent complex. The interaction element facilitates theformation of a bioluminescent complex by any suitable mechanism,including: interacting with one or both non-luminescent elements,inducing a conformational change in a non-luminescent element,interacting with another interaction element (e.g., an interactionelement attached to the other non-luminescent element), bringingnon-luminescent elements into close proximity, orienting non-luminescentelements for proper interaction, etc.

In some embodiments, one or more interaction elements are added to asolution containing the non-luminescent elements, but are not attachedto the non-luminescent elements. In such embodiments, the interactionelement(s) interact with the non-luminescent elements to induceformation of the bioluminescent complex or create conditions suitablefor formation of the bioluminescent complex. In other embodiments, asingle interaction element is attached to one member of anon-luminescent pair. In such embodiments, the lone interaction elementinteracts with one or both of the non-luminescent elements to createfavorable interactions for formation of the bioluminescent complex. Intypical embodiments of the present invention, one interaction element isattached to each member of a non-luminescent pair. Favorableinteractions between the interaction elements facilitate interactionsbetween the non-luminescent elements. The interaction pair may stablyinteract, transiently interact, form a complex, etc. The interaction ofthe interaction pair facilitates interaction of the non-luminescentelements (and formation of a bioluminescent complex) by any suitablemechanism, including, but not limited to: bringing the non-luminescentpair members into close proximity, properly orienting thenon-luminescent pair members from interaction, reducing non-covalentforces acting against non-luminescent pair interaction, etc.

In some embodiments, an interaction pair comprises any two chemicalmoieties that facilitate interaction of an associated non-luminescentpair. An interaction pair may consist of, for example: two complementarynucleic acids, two polypeptides capable of dimerization (e.g.,homodimer, heterodimer, etc.), a protein and ligand, protein and smallmolecule, an antibody and epitope, a reactive pair of small molecules,etc. Any suitable pair of interacting molecules may find use as aninteraction pair.

In some embodiments, an interaction pair comprises two molecules ofinterest (e.g., proteins of interest) or target molecules. In someembodiments, compositions and methods herein provide useful assays(e.g., in vitro, in vivo, in situ, whole animal, etc.) for studying theinteractions between a pair of target molecules.

In certain embodiments, a pair off interaction elements, each attachedto one of the non-luminescent elements, interact with each other andthereby facilitate formation of the bioluminescent complex. In someembodiments, the presence of a ligand, substrate, co-factor or additioninteraction element (e.g., not attached to non-luminescent element) isnecessary to induce the interaction between the interaction elements andfacilitate bioluminescent complex formation. In some embodiments,detecting a signal from the bioluminescent complex indicates thepresence of the ligand, substrate, co-factor or addition interactionelement or conditions that allow for interaction with the interactionelements.

In some embodiments, a pair off interaction elements, and a pair ofnon-luminescent elements are all present in a single amino acid chain(e.g., (interaction element 1)-NLpep-(interaction element 2)-NLpoly,NLpoly-(interaction element 1)-NLpep-(interaction element 2),NLpoly-(interaction element 1)-(interaction element 2)-NLpep, etc.). Insome embodiments in which a pair off interaction elements, and a pair ofnon-luminescent elements are all present in a single amino acid chain, aligand, substrate, co-factor or addition interaction element is requiredfor the interaction pair to form an interaction complex and facilitateformation of the bioluminescent complex.

In certain embodiments, an interaction element and a non-luminescentelement are attached, fused, linked, connected, etc. In typicalembodiments, a first non-luminescent element and a first interactionelement are attached to each other, and a second non-luminescent elementand a second interaction element are attached to each other. Attachmentof signal and interaction elements may be achieved by any suitablemechanism, chemistry, linker, etc. The interaction and non-luminescentelements are typically attached through covalent connection, butnon-covalent linking of the two elements is also provided. In someembodiments, the signal and interaction elements are directly connectedand, in other embodiments, they are connected by a linker.

In some embodiments, in which the interaction element is a peptide orpolypeptide, the signal and interaction elements are contained within asingle amino acid chain. In some embodiments, a single amino acid chaincomprises, consists of, or consists essentially of a non-luminescentelement and interaction element. In some embodiments, a single aminoacid chain comprises, consists of, or consists essentially of anon-luminescent element, an interaction element, and optionally one ormore an N-terminal sequence, a C-terminal sequence, regulatory elements(e.g., promoter, translational start site, etc.), and a linker sequence.In some embodiments, the signal and interaction elements are containedwithin a fusion polypeptide. The signal and interaction elements (andany other amino acid segments to be included in the fusion) may beexpressed separately; however, in other embodiments, a fusion protein isexpressed that comprises or consist of both the interaction and signalsequences.

In some embodiments, a first fusion protein comprising a firstnon-luminescent element and first interaction element as well as asecond fusion protein comprising a second non-luminescent element andsecond interaction element are expressed within the same cells. In suchembodiments, the first and second fusion proteins are purified and/orisolated from the cells, or the interaction of the fusion proteins isassayed within the cells. In other embodiments, first and second fusionproteins are expressed in separate cells and combined (e.g., followingpurification and/or isolation, or following fusion of the cells orportions of the cells, or by transfer of a fusion protein from one cellto another, or by secretion of one or more fusion proteins into theextracellular medium) for signal detection. In some embodiments, one ormore fusion proteins are expressed in cell lysate (e.g., rabbitreticulocyte lysate) or in a cell-free system. In some embodiments, oneor more fusion proteins are expressed from the genome of a virus orother cellular pathogen.

In certain embodiments, nucleic acids, DNA, RNA, vectors, etc. areprovided that encode peptide, polypeptides, fusion polypeptide, fusionproteins, etc. of the present invention. Such nucleic acids and vectorsmay be used for expression, transformation, transfection, injection,etc.

In some embodiments, a non-luminescent element and interaction elementare connected by a linker. In some embodiments, a linker connects thesignal and interaction elements while providing a desired amount ofspace/distance between the elements. In some embodiments, a linkerallows both the signal and interaction elements to form their respectivepairs (e.g., non-luminescent pair and interaction pair) simultaneously.In some embodiments, a linker assists the interaction element infacilitating the formation of a non-luminescent pair interaction. Insome embodiments, when an interaction pair is formed, the linkers thatconnect each non-luminescent element to their respective interactionelements position the non-luminescent elements at the proper distanceand conformation to form a bioluminescent complex. In some embodiments,an interaction element and non-luminescent element are held in closeproximity (e.g., <4 monomer units) by a linker. In some embodiments, alinker provides a desired amount of distance (e.g., 1, 2, 3, 4, 5, 6 . .. 10 . . . 20, or more monomer units) between signal and interactionelements (e.g., to prevent undesirable interactions between signal andinteraction elements, for steric considerations, to allow properorientation of non-luminescent element upon formation of interactioncomplex, to allow propagation of a complex-formation from interactioncomplex to non-luminescent elements, etc.). In certain embodiments, alinker provides appropriate attachment chemistry between the signal andinteraction elements. A linker may also improve the synthetic process ofmaking the signal and interaction element (e.g., allowing them to besynthesized as a single unit, allowing post synthesis connection of thetwo elements, etc.).

In some embodiments, a linker is any suitable chemical moiety capable oflinking, connecting, or tethering a non-luminescent element to aninteraction element. In some embodiments, a linker is a polymer of oneor more repeating or non-repeating monomer units (e.g., nucleic acid,amino acid, carbon-containing polymer, carbon chain, etc.). When anon-luminescent element and interaction element are part of a fusionprotein, a linker (when present) is typically an amino acid chain. Whena non-luminescent element and interaction element are tethered togetherafter the expression of the individual elements, a linker may compriseany chemical moiety with functional (or reactive) groups at either endthat are reactive with functional groups on the signal and interactionelements, respectively. Any suitable moiety capable of tethering thesignal and interaction elements may find use as a linker.

A wide variety of linkers may be used. In some embodiments, the linkeris a single covalent bond. In some embodiments, the linker comprises alinear or branched, cyclic or heterocyclic, saturated or unsaturated,structure having 1-20 nonhydrogen atoms (e.g., C, N, P, O and S) and iscomposed of any combination of alkyl, ether, thioether, imine,carboxylic, amine, ester, carboxamide, sulfonamide, hydrazide bonds andaromatic or heteroaromatic bonds. In some embodiments, linkers arelonger than 20 nonhydrogen atoms (e.g. 21 non-hydrogen atoms, 25non-hydrogen atoms, 30 non-hydrogen atoms, 40 non-hydrogen atoms, 50non-hydrogen atoms, 100 non-hydrogen atoms, etc.) In some embodiments,the linker comprises 1-50 non-hydrogen atoms (in addition to hydrogenatoms) selected from the group of C, N, P, O and S (e.g. 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50 non-hydrogen atoms).

The present invention is not limited by the types of linkers available.The signal and interaction elements are linked, either directly (e.g.linker consists of a single covalent bond) or linked via a suitablelinker. The present invention is not limited to any particular linkergroup. A variety of linker groups are contemplated, and suitable linkerscould comprise, but are not limited to, alkyl groups, methylene carbonchains, ether, polyether, alkyl amide linker, a peptide linker, amodified peptide linker, a Poly(ethylene glycol) (PEG) linker, astreptavidin-biotin or avidin-biotin linker, polyaminoacids (e.g.polylysine), functionalised PEG, polysaccharides, glycosaminoglycans,dendritic polymers (WO93/06868 and by Tomalia et al. in Angew. Chem.Int. Ed. Engl. 29:138-175 (1990), herein incorporated by reference intheir entireties), PEG-chelant polymers (W94/08629, WO94/09056 andWO96/26754, herein incorporated by reference in their entireties),oligonucleotide linker, phospholipid derivatives, alkenyl chains,alkynyl chains, disulfide, or a combination thereof. In someembodiments, the linker is cleavable (e.g., enzymatically (e.g., TEVprotease site), chemically, photoinduced, etc.

In some embodiments, substantially non-luminescent peptides andpolypeptides are provided with less than 100% sequence identity and/orsimilarity to any portion of an existing luciferase (e.g., a fireflyluciferase, a Renilla luciferase, an Oplophorus luciferase, enhancedOplophorus luciferases as described in U.S. Pat. App. 2010/0281552 andU.S. Pat. App. 2012/0174242, herein incorporated by reference in theirentireties). Certain embodiments of the present invention involve theformation of bioluminescent complexes of non-luminescent peptides andpolypeptides with less than 100% sequence identity with all or a portion(e.g., 8 or more amino acids, less than about 25 amino acids forpeptides) of SEQ ID NO: 2157 (e.g., complete NANOLUC sequence). Certainembodiments of the present invention involve the formation ofbioluminescent complexes of non-luminescent peptides and polypeptideswith less than 100%, but more than 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity with all or a portion (e.g., 8 or more amino acids,less than about 25 amino acids for peptides) of SEQ ID NO: 2157 (e.g.,complete NANOLUC sequence). In some embodiments, non-luminescentpeptides and polypeptides are provided with less than 100% sequencesimilarity with a portion (e.g., 8 or more amino acids, less than about25 amino acids for peptides) of SEQ ID NO: 2157 (e.g., peptides andpolypeptides that interact to form bioluminescent complexes). In someembodiments, non-luminescent peptides and polypeptides are provided withless than 100%, but more than 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence similarity with a portion (e.g., 8 or more amino acids, lessthan about 25 amino acids for peptides) of SEQ ID NO: 2157 (e.g.,peptides and polypeptides that interact to form bioluminescentcomplexes). Non-luminescent peptides are provided that have less than100% sequence identity and/or similarity with about a 25 amino acid orless portion of SEQ ID NO: 2157, wherein such peptides form abioluminescent complex when combined under appropriate conditions (e.g.,stabilized by an interaction pair) with a polypeptide having less than100%, but more than 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity and/or similarity with another portion SEQ ID NO:2157. Non-luminescent peptides are provided that have less than 100%sequence identity and/or similarity with about a 25 amino acid or lessportion of SEQ ID NO: 2157, wherein such peptides form a bioluminescentcomplex when combined under appropriate conditions (e.g., stabilized byan interaction pair) with a polypeptide having less than 100%, but morethan 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity and/or similarity with another portion SEQ ID NO:2157. Non-luminescent peptides are provided that have less than 100%,but more than 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity and/or similarity with about a 25 amino acid or lessportion of SEQ ID NO: 2157, wherein such peptides form a bioluminescentcomplex when combined under appropriate conditions (e.g., stabilized byan interaction pair) with a polypeptide having less than 100%, but morethan 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity and/or similarity with another portion SEQ ID NO:2157. Similarly, non-luminescent polypeptides are provided that haveless than 100%, but more than 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity or similarity with a portion of SEQ ID NO: 2157,wherein such polypeptides form a bioluminescent complex when combinedunder appropriate conditions (e.g., stabilized by an interaction pair)with a peptide having less than 100%, but optionally more than 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity and/or similarity with another portion SEQ ID NO:2157. In some embodiments, non-luminescent peptides with less than 100sequence identity or similarity with SEQ ID NO: 2 are provided. In someembodiments, non-luminescent peptides with less than 100%, but more than40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity or similarity with SEQ ID NO: 2 are provided. In someembodiments, non-luminescent polypeptides with less than 100 sequenceidentity or similarity with SEQ ID NO: 440 are provided.

In some embodiments, non-luminescent polypeptides with less than 100%,but more than 40%(e.g., >40%, >45%, >50%, >55%, >60%, >65%, >70%, >75%, >80%, >85%, >90%, >95%, >98%, >99%)sequence identity or similarity with SEQ ID NO: 440 are provided.

In some embodiments, non-luminescent peptides that find use inembodiments of the present invention include peptides with one or moreamino acid substitutions, deletions, or additions from GVTGWRLCKRISA(SEQ ID NO: 236). In some embodiments, the present invention providespeptides comprising one or more of amino acid sequences of Table 1,and/or nucleic acids comprising the nucleic acid sequences of Table 1(which code for the peptide sequences of Table 1).

TABLE 1  Peptide sequences SEQ ID NO. PEPTIDE NO. POLYMER SEQUENCE 3NLpep2 (w/ Met) N.A. ATGGACGTGACCGGCTGGCGGCTGTGCGAACGCATTCT GGCG 4NLpep2 (w/ Met) A.A. MDVTGWRLCERILA 5 NLpep3 (w/ Met) N.A.ATGGGAGTGACCGCCTGGCGGCTGTGCGAACGCATTCT GGCG 6 NLpep3 (w/ Met) A.A.MGVTAWRLCERILA 7 NLpep4 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTC TGGCG 8 NLpep4 (w/ Met) A.A.MGVTGWRLCKRILA 9 NLpep5 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTA GCGCG 10 NLpep5 (w/ Met) A.A.MGVTGWRLCERISA 11 NLpep6 (w/ Met) N.A.ATGGACGTGACCGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 12 NLpep6 (w/ Met) A.A.MDVTGWRLCKRISA 13 NLpep7 (w/ Met) N.A.ATGGACGTGACCGGCTGGCGGCTGTGCAAGCGCATTCT GGCG 14 NLpep7 (w/ Met) A.A.MDVTGWRLCKRILA 15 NLpep8 (w/ Met) N.A.ATGGACGTGACCGGCTGGCGGCTGTGCGAACGCATTA GCGCG 16 NLpep8 (w/ Met) A.A.MDVTGWRLCERISA 17 NLpep9 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 18 NLpep9 (w/ Met) A.A.MGVTGWRLCKRISA 19 NLpep10 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGAACGAACGCATTC TGGCG 20 NLpep10 (w/ Met) A.A.MGVTGWRLNERILA 21 NLpep11 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGCAGGAACGCATTC TGGCG 22 NLpep11 (w/ Met) A.A.MGVTGWRLQERILA 23 NLpep12 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGAAGAAGCGCCGGA GCCGG 24 NLpep12 (w/ Met) A.A.MGVTGWRLKKRRSR 25 NLpep13 (w/ Met) N.A.ATGAACGTGACCGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 26 NLpep13 (w/ Met) A.A.MNVTGWRLCKRISA 27 NLpep14 (w/ Met) N.A.ATGAGCGTGACCGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 28 NLpep14 (w/ Met) A.A.MSVTGWRLCKRISA 29 NLpep15 (w/ Met) N.A.ATGGAGGTGACCGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 30 NLpep15 (w/ Met) A.A.MEVTGWRLCKRISA 31 NLpep16 (w/ Met) N.A.ATGGGCGTGACCGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 32 NLpep16 (w/ Met) A.A.MHVTGWRLCKRISA 33 NLpep17 (w/ Met) N.A.ATGGGACACACCGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 34 NLpep17 (w/ Met) A.A.MGITGWRLCKRISA 35 NLpep18 (w/ Met) N.A.ATGGGAGCCACCGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 36 NLpep18 (w/ Met) A.A.MGATGWRLCKRISA 37 NLpep19 (w/ Met) N.A.ATGGGAAAGACCGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 38 NLpep19 (w/ Met) A.A.MGKTGWRLCKRISA 39 NLpep20 (w/ Met) N.A.ATGGGACAGACCGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 40 NLpep20 (w/ Met) A.A.MGQTGWRLCKRISA 41 NLpep21 (w/ Met) N.A.ATGGGAAGCACCGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 42 NLpep21 (w/ Met) A.A.MGSTGWRLCKRISA 43 NLpep22 (w/ Met) N.A.ATGGGAGTGGTGGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 44 NLpep22 (w/ Met) A.A.MGVVGWRLCKRISA 45 NLpep23 (w/ Met) N.A.ATGGGAGTGAAGGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 46 NLpep23 (w/ Met) A.A.MGVKGWRLCKRISA 47 NLpep24 (w/ Met) N.A.ATGGGAGTGCAGGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 48 NLpep24 (w/ Met) A.A.MGVQGWRLCKRISA 49 NLpep25 (w/ Met) N.A.ATGGGAGTGACCGGCACCCGGCTGTGCAAGCGCATTA GCGCG 50 NLpep25 (w/ Met) A.A.MGVTGTRLCKRISA 51 NLpep26 (w/ Met) N.A.ATGGGAGTGACCGGCAAGCGGCTGTGCAAGCGCATTA GCGCG 52 NLpep26 (w/ Met) A.A.MGVTGKRLCKRISA 53 NLpep27 (w/ Met) N.A.ATGGGAGTGACCGGCGTGCGGCTGTGCAAGCGCATTA GCGCG 54 NLpep27 (w/ Met) A.A.MGVTGVRLCKRISA 55 NLpep28 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCACTGCAAGCGCATTA GCGCG 56 NLpep28 (w/ Met) A.A.MGVTGWRICKRISA 57 NLpep29 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGGTGTGCAAGCGCATTA GCGCG 58 NLpep29 (w/ Met) A.A.MGVTGWRVCKRISA 59 NLpep30 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGACCTGCAAGCGCATTA GCGCG 60 NLpep30 (w/ Met) A.A.MGVTGWRTCKRISA 61 NLpep31 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGTACTGCAAGCGCATTA GCGCG 62 NLpep31 (w/ Met) A.A.MGVTGWRYCKRISA 63 NLpep32 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGAAGTGCAAGCGCATTA GCGCG 64 NLpep32 (w/ Met) A.A.MGVTGWRKCKRISA 65 NLpep33 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGAACAAGCGCATTA GCGCG 66 NLpep33 (w/ Met) A.A.MGVTGWRLNKRISA 67 NLpep34 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGACCAAGCGCATTA GCGCG 68 NLpep34 (w/ Met) A.A.MGVTGWRLTKRISA 69 NLpep35 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCAAGAAGATTA GCGCG 70 NLpep35 (w/ Met) A.A.MGVTGWRLCKKISA 71 NLpep36 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCAAGAACATTA GCGCG 72 NLpep36 (w/ Met) A.A.MGVTGWRLCKNISA 73 NLpep37 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCGTGA GCGCG 74 NLpep37 (w/ Met) A.A.MGVTGWRLCKRVSA 75 NLpep38 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCCAGA GCGCG 76 NLpep38 (w/ Met) A.A.MGVTGWRLCKRQSA 77 NLpep39 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCGAGA GCGCG 78 NLpep39 (w/ Met) A.A.MGVTGWRLCKRESA 79 NLpep40 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCCGGA GCGCG 80 NLpep40 (w/ Met) A.A.MGVTGWRLCKRRSA 81 NLpep41 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCTTCA GCGCG 82 NLpep41 (w/ Met) A.A.MGVTGWRLCKRFSA 83 NLpep42 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTA GCAAC 84 NLpep42 (w/ Met) A.A.MGVTGWRLCKRISN 85 NLpep43 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTA GCACC 86 NLpep43 (w/ Met) A.A.MGVTGWRLCKRIST 87 NLpep44 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTA GCCGG 88 NLpep44 (w/ Met) A.A.MGVTGWRLCKRISR 89 NLpep45 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTA GCCTG 90 NLpep45 (w/ Met) A.A.MGVTGWRLCKRISL 91 NLpep46 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTGCAAGCGCATTA GCGAG 92 NLpep46 (w/ Met) A.A.MGVTGWRLCKRISE 93 NLpep47 (w/ Met) N.A.ATGGGAGTGACCGGCTTCCGGCTGTGCAAGCGCATTA GCGCG 94 NLpep47 (w/ Met) A.A.MGVTGFRLCKRISA 95 NLpep48 (w/ Met) N.A.ATGGGAGTGACCGGCTACCGGCTGTGCAAGCGCATTA GCGCG 96 NLpep48 (w/ Met) A.A.MGVTGYRLCKRISA 97 NLpep49 (w/ Met) N.A.ATGGGAGTGACCGGCGAGCGGCTGTGCAAGCGCATTA GCGCG 98 NLpep49 (w/ Met) A.A.MGVTGERLCKRISA 99 NLpep50 (w/ Met) N.A.ATGCAGGTGACCGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 100 NLpep50 (w/ Met) A.A.MQVTGWRLCKRISA 101 NLpep51 (w/ Met) N.A.ATGACCGTGACCGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 102 NLpep51 (w/ Met) A.A.MTVTGWRLCKRISA 103 NLpep52 (w/ Met) N.A.ATGGGAGTGGAGGGCTGGCGGCTGTGCAAGCGCATTA GCGCG 104 NLpep52 (w/ Met) A.A.MGVEGWRLCKRISA 105 NLpep53 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTTCAAGCGCATTA GCGCG 106 NLpep53 (w/ Met) A.A.MGVTGWRLFKRISA 107 NLpep54 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTACAAGCGCATTA GCGCG 108 NLpep54 (w/ Met) A.A.MGVTGWRLYKRISA 109 NLpep55 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGAGCAAGCGCATTA GCGCG 110 NLpep55 (w/ Met) A.A.MGVTGWRLSKRISA 111 NLpep56 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGGGCAAGCGCATTA GCGCG 112 NLpep56 (w/ Met) A.A.MGVTGWRLHKRISA 113 NLpep57 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGATGAAGCGCATTA GCGCG 114 NLpep57 (w/ Met) A.A.MGVTGWRLMKRISA 115 NLpep58 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGGCCAAGCGCATTA GCGCG 116 NLpep58 (w/ Met) A.A.MGVTGWRLAKRISA 117 NLpep59 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGCAGAAGCGCATTA GCGCG 118 NLpep59 (w/ Met) A.A.MGVTGWRLQKRISA 119 NLpep60 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGCTGAAGCGCATTA GCGCG 120 NLpep60 (w/ Met) A.A.MGVTGWRLLKRISA 121 NLpep61 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGAAGAAGCGCATTA GCGCG 122 NLpep61 (w/ Met) A.A.MGVTGWRLKKRISA 123 NLpep62 (w/ Met) N.A.ATGAACCACACCGGCTGGCGGCTGAACAAGAAGGTGA GCAAC 124 NLpep62 (w/ Met) A.A.MNITGWRLNKKVSN 125 NLpep63 (w/ Met) N.A.ATGAACCACACCGGCTACCGGCTGAACAAGAAGGTGA GCAAC 126 NLpep63 (w/ Met) A.A.MNITGYRLNKKVSN 127 NLpep64 (w/ Met) N.A.ATGTGCGTGACCGGCTGGCGGCTGTTCAAGCGCATTAG CGCG 128 NLpep64 (w/ Met) A.A.MCVTGWRLFKRISA 129 NLpep65 (w/ Met) N.A.ATGCCCGTGACCGGCTGGCGGCTGTTCAAGCGCATTAG CGCG 130 NLpep65 (w/ Met) A.A.MPVTGWRLFKRISA 131 NLpep66 (w/ Met) N.A.ATGAACCACACCGGCTACCGGCTGTTCAAGAAGGTGA GCAAC 132 NLpep66 (w/ Met) A.A.MNITGYRLFKKVSN 133 NLpep67 (w/ Met) N.A.ATGAACGTGACCGGCTACCGGCTGTTCAAGAAGGTGA GCAAC 134 NLpep67 (w/ Met) A.A.MNVTGYRLFKKVSN 135 NLpep68 (w/ Met) N.A.ATGAACGTGACCGGCTGGCGGCTGTTCAAGAAGGTGA GCAAC 136 NLpep68 (w/ Met) A.A.MNVTGWRLFKKVSN 137 NLpep69 (w/ Met) N.A.ATGAACGTGACCGGCTGGCGGCTGTTCAAGAAGATTA GCAAC 138 NLpep69 (w/ Met) A.A.MNVTGWRLFKKISN 139 NLpep70 (w/ Met) N.A.ATGAACGTGACCGGCTGGCGGCTGTTCAAGCGCATTA GCAAC 140 NLpep70 (w/ Met) A.A.MNVTGWRLFKRISN 141 NLpep71 (w/ Met) N.A.ATGGGAGTGACCGGCTGGCGGCTGTTCAAGCGCATTA GCAAC 142 NLpep71 (w/ Met) A.A.MGVTGWRLFKRISN 143 NLpep72 (w/ Met) N.A.ATGAACGTGACCGGCTGGCGGCTGTTCGAACGCATTA GCAAC 144 NLpep72 (w/ Met) A.A.MNVTGWRLFERISN 145 NLpep73 (w/ Met) N.A.ATGAACGTGACCGGCTGGCGGCTGTTCAAGCGCATTCT GAAC 146 NLpep73 (w/ Met) A.A.MNVTGWRLFKRILN 147 NLpep74 (w/ Met) N.A.ATGAACGTGACCGGCTGGCGGCTGTTCAAGCGCATTA GCGCG 148 NLpep74 (w/ Met) A.A.MNVTGWRLFKRISA 149 NLpep75 (w/ Met) N.A.ATGAACGTGACCGGCTGGCGGCTGTTCGAAAAGATTA GCAAC 150 NLpep75 (w/ Met) A.A.MNVTGWRLFEKISN 151 NLpep76 (w/ Met) N.A.ATGAACGTGAGCGGCTGGCGGCTGTTCGAAAAGATTA GCAAC 152 NLpep76 (w/ Met) A.A.MNVSGWRLFEKISN 153 NLpep77 (w/ Met) N.A.ATG-GTGACCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 154 NLpep77 (w/ Met) A.A.M-VTGWRLFKKISN 155 NLpep78 (w/ Met) N.A.ATGAACGTGAGCGGCTGGCGGCTGTTCAAGAAGATTA GCAAC 156 NLpep78 (w/ Met) A.A.MNVSGWRLFKKISN 157 NLpep79 (w/ Met) N.A.ATGAACGTGACCGGCTACCGGCTGTTCAAGAAGATTA GCAAC 158 NLpep79 (w/ Met) A.A.MNVTGYRLFKKISN 159 NLpep80 (w/ Met) N.A.ATGGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCA AC 160 NLpep80 (w/ Met) A.A.MVSGWRLFKKISN 161 NLpep81 (w/ Met) N.A.ATGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCAAC 440 NLpep81 (w/ Met) A.A.MSGWRLFKKISN 163 NLpep82 (w/ Met) N.A. ATGGGCTGGCGGCTGTTCAAGAAGATTAGCAAC164 NLpep82 (w/ Met) A.A. MGWRLFKKISN 165 NLpep83 (w/ Met) N.A.ATGAACGTGAGCGGCTGGCGGCTGTTCAAGAAGATTA GC 166 NLpep83 (w/ Met) A.A.MNVSGWRLFKKIS 167 NLpep84 (w/ Met) N.A.ATGAACGTGAGCGGCTGGCGGCTGTTCAAGAAGATT 168 NLpep84 (w/ Met) A.A.MNVSGWRLFKKI 169 NLpep85 (w/ Met) N.A. ATGAACGTGAGCGGCTGGCGGCTGTTCAAGAAG170 NLpep85 (w/ Met) A.A. MNVSGWRLFKK 171 NLpep86 (w/ Met) N.A.ATGGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGC 172 NLpep86 (w/ Met) A.A.MVSGWRLFKKIS 173 NLpep87 (w/ Met) N.A. ATGAGCGGCTGGCGGCTGTTCAAGAAGATT174 NLpep87 (w/ Met) A.A. MSGWRLFKKI 175 NLpep88 (w/ Met) N.A.ATGAACGTGAGCGGCTGGGGCCTGTTCAAGAAGATTA GCAAC

In certain embodiments, a peptide from Table 1 is provided. In someembodiments, peptides comprise a single amino acid difference fromGVTGWRLCKRISA (SEQ ID NO: 236) and/or any of the peptides listed inTable 1. In some embodiments, peptides comprise two or more (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10, etc.) amino acid differences from GVTGWRLCKRISA(SEQ ID NO: 236) and/or any of the peptides listed in Table 1. In someembodiments, peptides are provided comprising one of the amino acidsequences of SEQ ID NOS: 3-438 and 2162-2365. In some embodiments,peptides are provided comprising one of the amino acid sequences of SEQID NOS: 3-438 and 2162-2365 with one or more additions, substitutions,and/or deletions. In some embodiments, a peptide or a portion thereofcomprises greater than 70% sequence identity (e.g., 71%, 75%, 80%, 85%,90%, 95%, 99%) with one or more of the amino acid sequence of SEQ IDNOS: 3-438 and 2162-2365. In some embodiments, nucleic acids areprovided comprising one of the nucleic acid coding sequences of SEQ IDNOS: 3-438 and 2162-2365. In some embodiments, nucleic acids areprovided comprising one of the nucleic acid sequences of SEQ ID NOS:3-438 and 2162-2365 with one or more additions, substitutions, and/ordeletions. In some embodiments, a nucleic acid or a portion thereofcomprises greater than 70% sequence identity (e.g., 71%, 75%, 80%, 85%,90%, 95%, 99%) with one or more of the nucleic acid sequence of SEQ IDNOS: 3-438 and 2162-2365. In some embodiments, nucleic acids areprovided that code for one of the amino acid sequences of SEQ ID NOS:3-438 and 2162-2365. In some embodiments, nucleic acids are providedthat code for one of the amino acid sequences of SEQ ID NOS: 3-438 and2162-2365 with one or more additions, substitutions, and/or deletions.In some embodiments, a nucleic acid is provided that codes for an aminoacid with greater than 70% sequence identity (e.g., 71%, 75%, 80%, 85%,90%, 95%, 99%) with one or more of the amino acid sequences of SEQ IDNOS: 3-438 and 2162-2365.

In certain embodiments, a nucleic acid from Table 1 is provided. In someembodiments, a nucleic acid encoding a peptide from Table 1 is provided.In some embodiments, a nucleic acid of the present invention codes for apeptide that comprises a single amino acid difference fromMGVTGWRLCERILA (SEQ ID NO: 2) and/or any of the peptides listed inTable 1. In some embodiments, nucleic acids code for peptides comprisingtwo or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino aciddifferences from MGVTGWRLCERILA (SEQ ID NO: 2) and/or any of thepeptides listed in Table 1. In some embodiments, nucleic acids areprovided comprising the sequence of one of the nucleic acids in Table 1.In some embodiments, nucleic acids are provided comprising one of thenucleic acids of Table 1 with one or more additions, substitutions,and/or deletions. In some embodiments, a nucleic acid or a portionthereof comprises greater than 70% sequence identity (e.g., 71%, 75%,80%, 85%, 90%, 95%, 99%) with one or more of the nucleic acids of Table1.

In some embodiments, non-luminescent polypeptides that find use inembodiments of the present invention include polypeptides with one ormore amino acid substitutions, deletions, or additions from SEQ ID NO:440. In some embodiments, the present invention provides polypeptidescomprising one or more of amino acid sequences of Table 2, and/ornucleic acids comprising the nucleic acid sequences of Table 2 (whichcode for the polypeptide sequences of Table 2).

TABLE 2 Polypeptide sequences SEQ SEQ SEQ ID NO Polymer ID ID NO Poly.ID ID NO Poly. ID 441 N.A. R11N 727 N.A. 5A2 + V58P 1013 N.A. 5P D6(−152-157) 442 A.A R11N 728 A.A 5A2 + V58P 1014 A.A 5P D6 (−152-157) 443N.A. T13I 729 N.A. 5A2 + V58Q 1015 N.A. 5P D7 (−151-157) 444 A.A T13I730 A.A 5A2 + V58Q 1016 A.A 5P D7 (−151-157) 445 N.A. G15S 731 N.A.5A2 + V58R 1017 N.A. 5P + F31A 446 A.A G15S 732 A.A 5A2 + V58R 1018 A.A5P + F31A 447 N.A. L18Q 733 N.A. 5A2 + V58S 1019 N.A. 5P + F31C 448 A.AL18Q 734 A.A 5A2 + V58S 1020 A.A 5P + F31C 449 N.A. Q20K 735 N.A. 5A2 +V58T 1021 N.A. 5P + F31D 450 A.A Q20K 736 A.A 5A2 + V58T 1022 A.A 5P +F31D 451 N.A. V27M 737 N.A. 5A2 + V58W 1023 N.A. 5P + F31E 452 A.A V27M738 A.A 5A2 + V58W 1024 A.A 5P + F31E 453 N.A. F31I 739 N.A. 5A2 + V58Y1025 N.A. 5P + F31G 454 A.A F31I 740 A.A 5A2 + V58Y 1026 A.A 5P + F31G455 N.A. F31L 741 N.A. 5A2 + A67C 1027 N.A. 5P + F31H 456 A.A F31L 742A.A 5A2 + A67C 1028 A.A 5P + F31H 457 N.A. F31V 743 N.A. 5A2 + A67D 1029N.A. 5P + F31I 458 A.A F31V 744 A.A 5A2 + A67D 1030 A.A 5P + F31I 459N.A. Q32R 745 N.A. 5A2 + A67E 1031 N.A. 5P + F31K 460 A.A Q32R 746 A.A5A2 + A67E 1032 A.A 5P + F31K 461 N.A. N33K 747 N.A. 5A2 + A67F 1033N.A. 5P + F31L 462 A.A N33K 748 A.A 5A2 + A67F 1034 A.A 5P + F31L 463N.A. N33R 749 N.A. 5A2 + A67G 1035 N.A. 5P + F31M 464 A.A N33R 750 A.A5A2 + A67G 1036 A.A 5P + F31M 465 N.A. I56N 751 N.A. 5A2 + A67H 1037N.A. 5P + F31N 466 A.A I56N 752 A.A 5A2 + A67H 1038 A.A 5P + F31N 467N.A. V58A 753 N.A. 5A2 + A67I 1039 N.A. 5P + F31P 468 A.A V58A 754 A.A5A2 + A67I 1040 A.A 5P + F31P 469 N.A. I59T 755 N.A. 5A2 + A67K 1041N.A. 5P + F31Q 470 A.A I59T 756 A.A 5A2 + A67K 1042 A.A 5P + F31Q 471N.A. G67S 757 N.A. 5A2 + A67L 1043 N.A. 5P + F31R 472 A.A G67S 758 A.A5A2 + A67L 1044 A.A 5P + F31R 473 N.A. G67D 759 N.A. 5A2 + A67M 1045N.A. 5P + F31S 474 A.A G67D 760 A.A 5A2 + A67M 1046 A.A 5P + F31S 475N.A. K75E 761 N.A. 5A2 + A67N 1047 N.A. 5P + F31T 476 A.A K75E 762 A.A5A2 + A67N 1048 A.A 5P + F31T 477 N.A. M106V 763 N.A. 5A2 + A67P 1049N.A. 5P + F31V 478 A.A M106V 764 A.A 5A2 + A67P 1050 A.A 5P + F31V 479N.A. M106I 765 N.A. 5A2 + A67Q 1051 N.A. 5P + F31W 480 A.A M106I 766 A.A5A2 + A67Q 1052 A.A 5P + F31W 481 N.A. D108N 767 N.A. 5A2 + A67R 1053N.A. 5P + F31Y 482 A.A D108N 768 A.A 5A2 + A67R 1054 A.A 5P + F31Y 483N.A. R112Q 769 N.A. 5A2 + A67S 1055 N.A. 5P + L46A 484 A.A R112Q 770 A.A5A2 + A67S 1056 A.A 5P + L46A 485 N.A. N144T 771 N.A. 5A2 + A67T 1057N.A. 5P + L46C 486 A.A N144T 772 A.A 5A2 + A67T 1058 A.A 5P + L46C 487N.A. L149M 773 N.A. 5A2 + A67V 1059 N.A. 5P + L46D 488 A.A L149M 774 A.A5A2 + A67V 1060 A.A 5P + L46D 489 N.A. N156D 775 N.A. 5A2 + A67W 1061N.A. 5P + L46E 490 A.A N156D 776 A.A 5A2 + A67W 1062 A.A 5P + L46E 491N.A. N156S 777 N.A. 5A2 + A67Y 1063 N.A. 5P + L46F 492 A.A N156S 778 A.A5A2 + A67Y 1064 A.A 5P + L46F 493 N.A. V157D 779 N.A. 5A2 + M106A 1065N.A. 5P + L46G 494 A.A V157D 780 A.A 5A2 + M106A 1066 A.A 5P + L46G 495N.A. V157S 781 N.A. 5A2 + M106C 1067 N.A. 5P + L46H 496 A.A V157S 782A.A 5A2 + M106C 1068 A.A 5P + L46H 497 N.A. G8A 783 N.A. 5A2 + M106D1069 N.A. 5P + L46I 498 A.A G8A 784 A.A 5A2 + M106D 1070 A.A 5P + L46I499 N.A. G15A 785 N.A. 5A2 + M106E 1071 N.A. 5P + L46K 500 A.A G15A 786A.A 5A2 + M106E 1072 A.A 5P + L46K 501 N.A. G25A 787 N.A. 5A2 + M106F1073 N.A. 5P + L46M 502 A.A G25A 788 A.A 5A2 + M106F 1074 A.A 5P + L46M503 N.A. G26A 789 N.A. 5A2 + M106G 1075 N.A. 5P + L46N 504 A.A G26A 790A.A 5A2 + M106G 1076 A.A 5P + L46N 505 N.A. G35A 791 N.A. 5A2 + M106H1077 N.A. 5P + L46P 506 A.A G35A 792 A.A 5A2 + M106H 1078 A.A 5P + L46P507 N.A. G48A 793 N.A. 5A2 + M106I 1079 N.A. 5P + L46Q 508 A.A G48A 794A.A 5A2 + M106I 1080 A.A 5P + L46Q 509 N.A. G51A 795 N.A. 5A2 + M106K1081 N.A. 5P + L46R 510 A.A G51A 796 A.A 5A2 + M106K 1082 A.A 5P + L46R511 N.A. G64A 797 N.A. 5A2 + M106L 1083 N.A. 5P + L46S 512 A.A G64A 798A.A 5A2 + M106L 1084 A.A 5P + L46S 513 N.A. G67A 799 N.A. 5A2 + M106N1085 N.A. 5P + L46T 514 A.A G67A 800 A.A 5A2 + M106N 1086 A.A 5P + L46T515 N.A. G71A 801 N.A. 5A2 + M106P 1087 N.A. 5P + L46V 516 A.A G71A 802A.A 5A2 + M106P 1088 A.A 5P + L46V 517 N.A. G95A 803 N.A. 5A2 + M106Q1089 N.A. 5P + L46W 518 A.A G95A 804 A.A 5A2 + M106Q 1090 A.A 5P + L46W519 N.A. G101A 805 N.A. 5A2 + M106R 1091 N.A. 5P + L46Y 520 A.A G101A806 A.A 5A2 + M106R 1092 A.A 5P + L46Y 521 N.A. G111A 807 N.A. 5A2 +M106S 1093 N.A. 5P + N108A 522 A.A G111A 808 A.A 5A2 + M106S 1094 A.A5P + N108A 523 N.A. G116A 809 N.A. 5A2 + M106T 1095 N.A. 5P + N108C 524A.A G116A 810 A.A 5A2 + M106T 1096 A.A 5P + N108C 525 N.A. G122A 811N.A. 5A2 + M106V 1097 N.A. 5P + N108D 526 A.A G122A 812 A.A 5A2 + M106V1098 A.A 5P + N108D 527 N.A. G129A 813 N.A. 5A2 + M106W 1099 N.A. 5P +N108E 528 A.A G129A 814 A.A 5A2 + M106W 1100 A.A 5P + N108E 529 N.A.G134A 815 N.A. 5A2 + M106Y 1101 N.A. 5P + N108F 530 A.A G134A 816 A.A5A2 + M106Y 1102 A.A 5P + N108F 531 N.A. G147A 817 N.A. 5A2 + L149A 1103N.A. 5P + N108G 532 A.A G147A 818 A.A 5A2 + L149A 1104 A.A 5P + N108G533 N.A. I54A 819 N.A. 5A2 + L149C 1105 N.A. 5P + N108H 534 A.A I54A 820A.A 5A2 + L149C 1106 A.A 5P + N108H 535 N.A. 5A1 821 N.A. 5A2 + L149D1107 N.A. 5P + N108I (G15A/D19A/ G35A/G51A/G67A) 536 A.A 5A1 822 A.A5A2 + L149D 1108 A.A 5P + N108I (G15A/D19A/ G35A/G51A/G67A) 537 N.A. 4A1823 N.A. 5A2 + L149E 1109 N.A. 5P + N108K (G15A/G35A/ G67A/G71A) 538 A.A4A1 824 A.A 5A2 + L149E 1110 A.A 5P + N108K (G15A/G35A/ G67A/G71A) 539N.A. 5A2 825 N.A. 5A2 + L149F 1111 N.A. 5P + N108L (G15A/G35A/G51A/G67A/G71A) 540 A.A 5A2 826 A.A 5A2 + L149F 1112 A.A 5P + N108L(G15A/G35A/ G51A/G67A/G71A) 541 N.A. 5A2 + A15G 827 N.A. 5A2 + L149G1113 N.A. 5P + N108M 542 A.A 5A2 + A15G 828 A.A 5A2 + L149G 1114 A.A5P + N108M 543 N.A. 5A2 + A35G 829 N.A. 5A2 + L149H 1115 N.A. 5P + N108P544 A.A 5A2 + A35G 830 A.A 5A2 + L149H 1116 A.A 5P + N108P 545 N.A.5A2 + A51G 831 N.A. 5A2 + L149I 1117 N.A. 5P + N108Q 546 A.A 5A2 + A51G832 A.A 5A2 + L149I 1118 A.A 5P + N108Q 547 N.A. 5A2 + A67G 833 N.A.5A2 + L149K 1119 N.A. 5P + N108R 548 A.A 5A2 + A67G 834 A.A 5A2 + L149K1120 A.A 5P + N108R 549 N.A. 5A2 + A71G 835 N.A. 5A2 + L149M 1121 N.A.5P + N108S 550 A.A 5A2 + A71G 836 A.A 5A2 + L149M 1122 A.A 5P + N108S551 N.A. 5A2 + R11A 837 N.A. 5A2 + L149N 1123 N.A. 5P + N108T 552 A.A5A2 + R11A 838 A.A 5A2 + L149N 1124 A.A 5P + N108T 553 N.A. 5A2 + R11C839 N.A. 5A2 + L149P 1125 N.A. 5P + N108V 554 A.A 5A2 + R11C 840 A.A5A2 + L149P 1126 A.A 5P + N108V 555 N.A. 5A2 + R11D 841 N.A. 5A2 + L149Q1127 N.A. 5P + N108W 556 A.A 5A2 + R11D 842 A.A 5A2 + L149Q 1128 A.A5P + N108W 557 N.A. 5A2 + R11E 843 N.A. 5A2 + L149R 1129 N.A. 5P + N108Y558 A.A 5A2 + R11E 844 A.A 5A2 + L149R 1130 A.A 5P + N108Y 559 N.A.5A2 + R11F 845 N.A. 5A2 + L149S 1131 N.A. 5P + T144A 560 A.A 5A2 + R11F846 A.A 5A2 + L149S 1132 A.A 5P + T144A 561 N.A. 5A2 + R11G 847 N.A.5A2 + L149T 1133 N.A. 5P + T144C 562 A.A 5A2 + R11G 848 A.A 5A2 + L149T1134 A.A 5P + T144C 563 N.A. 5A2 + R11H 849 N.A. 5A2 + L149V 1135 N.A.5P + T144D 564 A.A 5A2 + R11H 850 A.A 5A2 + L149V 1136 A.A 5P + T144D565 N.A. 5A2 + R11I 851 N.A. 5A2 + L149W 1137 N.A. 5P + T144E 566 A.A5A2 + R11I 852 A.A 5A2 + L149W 1138 A.A 5P + T144E 567 N.A. 5A2 + R11K853 N.A. 5A2 + L149Y 1139 N.A. 5P + T144F 568 A.A 5A2 + R11K 854 A.A5A2 + L149Y 1140 A.A 5P + T144F 569 N.A. 5A2 + R11L 855 N.A. 5A2 + V157A1141 N.A. 5P + T144G 570 A.A 5A2 + R11L 856 A.A 5A2 + V157A 1142 A.A5P + T144G 571 N.A. 5A2 + R11M 857 N.A. 5A2 + V157C 1143 N.A. 5P + T144H572 A.A 5A2 + R11M 858 A.A 5A2 + V157C 1144 A.A 5P + T144H 573 N.A.5A2 + R11N 859 N.A. 5A2 + V157D 1145 N.A. 5P + T144I 574 A.A 5A2 + R11N860 A.A 5A2 + V157D 1146 A.A 5P + T144I 575 N.A. 5A2 + R11P 861 N.A.5A2 + V157E 1147 N.A. 5P + T144K 576 A.A 5A2 + R11P 862 A.A 5A2 + V157E1148 A.A 5P + T144K 577 N.A. 5A2 + R11Q 863 N.A. 5A2 + V157F 1149 N.A.5P + T144L 578 A.A 5A2 + R11Q 864 A.A 5A2 + V157F 1150 A.A 5P + T144L579 N.A. 5A2 + R11S 865 N.A. 5A2 + V157G 1151 N.A. 5P + T144M 580 A.A5A2 + R11S 866 A.A 5A2 + V157G 1152 A.A 5P + T144M 581 N.A. 5A2 + R11T867 N.A. 5A2 + V157H 1153 N.A. 5P + T144N 582 A.A 5A2 + R11T 868 A.A5A2 + V157H 1154 A.A 5P + T144N 583 N.A. 5A2 + R11V 869 N.A. 5A2 + V157I1155 N.A. 5P + T144P 584 A.A 5A2 + R11V 870 A.A 5A2 + V157I 1156 A.A5P + T144P 585 N.A. 5A2 + R11W 871 N.A. 5A2 + V157K 1157 N.A. 5P + T144Q586 A.A 5A2 + R11W 872 A.A 5A2 + V157K 1158 A.A 5P + T144Q 587 N.A.5A2 + R11Y 873 N.A. 5A2 + V157L 1159 N.A. 5P + T144R 588 A.A 5A2 + R11Y874 A.A 5A2 + V157L 1160 A.A 5P + T144R 589 N.A. 5A2 + A15C 875 N.A.5A2 + V157M 1161 N.A. 5P + T144S 590 A.A 5A2 + A15C 876 A.A 5A2 + V157M1440 A.A 5P + T144S 591 N.A. 5A2 + A15D 877 N.A. 5A2 + V157N 1163 N.A.5P + T144V 592 A.A 5A2 + A15D 878 A.A 5A2 + V157N 1164 A.A 5P + T144V593 N.A. 5A2 + A15E 879 N.A. 5A2 + V157P 1165 N.A. 5P + T144W 594 A.A5A2 + A15E 880 A.A 5A2 + V157P 1166 A.A 5P + T144W 595 N.A. 5A2 + A15F881 N.A. 5A2 + V157Q 1167 N.A. 5P + T144Y 596 A.A 5A2 + A15F 882 A.A5A2 + V157Q 1168 A.A 5P + T144Y 597 N.A. 5A2 + A15G 883 N.A. 5A2 + V157R1169 N.A. 5P + P157A 598 A.A 5A2 + A15G 884 A.A 5A2 + V157R 1170 A.A5P + P157A 599 N.A. 5A2 + A15H 885 N.A. 5A2 + V157S 1171 N.A. 5P + P157C600 A.A 5A2 + A15H 886 A.A 5A2 + V157S 1172 A.A 5P + P157C 601 N.A.5A2 + A15I 887 N.A. 5A2 + V157T 1173 N.A. 5P + P157D 602 A.A 5A2 + A15I888 A.A 5A2 + V157T 1174 A.A 5P + P157D 603 N.A. 5A2 + A15K 889 N.A.5A2 + V157W 1175 N.A. 5P + P157E 604 A.A 5A2 + A15K 890 A.A 5A2 + V157W1176 A.A 5P + P157E 605 N.A. 5A2 + A15L 891 N.A. 5A2 + V157Y 1177 N.A.5P + P157F 606 A.A 5A2 + A15L 892 A.A 5A2 + V157Y 1178 A.A 5P + P157F607 N.A. 5A2 + A15M 893 N.A. 5A2 + Q20K 1179 N.A. 5P + P157G 608 A.A5A2 + A15M 894 A.A 5A2 + Q20K 1180 A.A 5P + P157G 609 N.A. 5A2 + A15N895 N.A. 5A2 + V27M 1181 N.A. 5P + P157H 610 A.A 5A2 + A15N 896 A.A5A2 + V27M 1182 A.A 5P + P157H 611 N.A. 5A2 + A15P 897 N.A. 5A2 + N33K1183 N.A. 5P + P157I 612 A.A 5A2 + A15P 898 A.A 5A2 + N33K 1184 A.A 5P +P157I 613 N.A. 5A2 + A15Q 899 N.A. 5A2 + V38I 1185 N.A. 5P + P157K 614A.A 5A2 + A15Q 900 A.A 5A2 + V38I 1186 A.A 5P + P157K 615 N.A. 5A2 +A15R 901 N.A. 5A2 + I56N 1187 N.A. 5P + P157L 616 A.A 5A2 + A15R 902 A.A5A2 + I56N 1188 A.A 5P + P157L 617 N.A. 5A2 + A15S 903 N.A. 5A2 + D108N1189 N.A. 5P + P157M 618 A.A 5A2 + A15S 904 A.A 5A2 + D108N 1190 A.A5P + P157M 619 N.A. 5A2 + A15T 905 N.A. 5A2 + N144T 1191 N.A. 5P + P157N620 A.A 5A2 + A15T 906 A.A 5A2 + N144T 1192 A.A 5P + P157N 621 N.A.5A2 + A15V 907 N.A. 5A2 + V27M + A35G 1193 N.A. 5P + P157Q 622 A.A 5A2 +A15V 908 A.A 5A2 + V27M + A35G 1194 A.A 5P + P157Q 623 N.A. 5A2 + A15W909 N.A. 5A2 + A71G + K75E 1195 N.A. 5P + P157R 624 A.A 5A2 + A15W 910A.A 5A2 + A71G + K75E 1196 A.A 5P + P157R 625 N.A. 5A2 + A15Y 911 N.A.5A2 + R11E + L149M 1197 N.A. 5P + P157S 626 A.A 5A2 + A15Y 912 A.A 5A2 +R11E + L149M 1198 A.A 5P + P157S 627 N.A. 5A2 + L18A 913 N.A. 5A2 +R11E + V157P 1199 N.A. 5P + P157T 628 A.A 5A2 + L18A 914 A.A 5A2 +R11E + V157P 1200 A.A 5P + P157T 629 N.A. 5A2 + L18C 915 N.A. 5A2 +D108N + N144T 1201 N.A. 5P + P157V 630 A.A 5A2 + L18C 916 A.A 5A2 +D108N + N144T 1202 A.A 5P + P157V 631 N.A. 5A2 + L18D 917 N.A. 5A2 +L149M + V157D 1203 N.A. 5P + P157W 632 A.A 5A2 + L18D 918 A.A 5A2 +L149M + V157D 1204 A.A 5P + P157W 633 N.A. 5A2 + L18E 919 N.A. 5A2 +L149M + V157P 1205 N.A. 5P + P157Y 634 A.A 5A2 + L18E 920 A.A 5A2 +L149M + V157P 1206 A.A 5P + P157Y 635 N.A. 5A2 + L18F 921 N.A. 3P 1207N.A. 5P + I107L (5A2 + R11E + L149M + V157P) 636 A.A 5A2 + L18F 922 A.A3P 1208 A.A 5P + I107L (5A2 + R11E + L149M + V157P) 637 N.A. 5A2 + L18G923 N.A. 3P + D108N 1209 N.A. 5P + K75E 638 A.A 5A2 + L18G 924 A.A 3P +D108N 1210 A.A 5P + K75E 639 N.A. 5A2 + L18H 925 N.A. 3P + N144T 1211N.A. 5P + K123E + N156D 640 A.A 5A2 + L18H 926 A.A 3P + N144T 1212 A.A5P + K123E + N156D 641 N.A. 5A2 + L18I 927 N.A. 3E 1213 N.A. 5P + I76V(5A2 + R11E + L149M + V157E) 642 A.A 5A2 + L18I 928 A.A 3E 1214 A.A 5P +I76V (5A2 + R11E + L149M + V157E) 643 N.A. 5A2 + L18K 929 N.A. 3E +D108N 1215 N.A. 5P + G48D + H57R + L92M + I99V 644 A.A 5A2 + L18K 930A.A 3E + D108N 1216 A.A 5P + G48D + H57R + L92M + I99V 645 N.A. 5A2 +L18M 931 N.A. 3E + N144T 1217 N.A. 5P + F31L + V36A + I99V 646 A.A 5A2 +L18M 932 A.A 3E + N144T 1218 A.A 5P + F31L + V36A + I99V 647 N.A. 5A2 +L18N 933 N.A. 5P 1219 N.A. 5P + F31L + H93P (3P + D108N + N144T) 648 A.A5A2 + L18N 934 A.A 5P 1220 A.A 5P + F31L + H93P (3P + D108N + N144T) 649N.A. 5A2 + L18P 935 N.A. 6P 1221 N.A. 5P + V90A (5P + I56N) 650 A.A5A2 + L18P 936 A.A 6P 1222 A.A 5P + V90A (5P + I56N) 651 N.A. 5A2 + L18Q937 N.A. 5E 1223 N.A. 5P + I44V (3E + D108N + N144T) 652 A.A 5A2 + L18Q938 A.A 5E 1224 A.A 5P + I44V (3E + D108N + N144T) 653 N.A. 5A2 + L18R939 N.A. 6E 1225 N.A. 5P + L46R + H86Q + (5E + I56N) M106V 654 A.A 5A2 +L18R 940 A.A 6E 1226 A.A 5P + L46R + H86Q + (5E + I56N) M106V 655 N.A.5A2 + L18S 941 N.A. NLpoly1 1227 N.A. 5P + R141H (5A2 + R11N + A15S +L18Q + F31I + V58A + A67D + M106V + L149M + V157D) 656 A.A 5A2 + L18S942 A.A NLpoly1 1228 A.A 5P + R141H (5A2 + R11N + A15S + L18Q + F31I +V58A + A67D + M106V + L149M + V157D) 657 N.A. 5A2 + L18T 943 N.A.NLpoly2 1229 N.A. 5P + N33D + V58A (5A2 + A15S + L18Q + F31I + V58A +A67D + M106V + L149M + V157D) 658 A.A 5A2 + L18T 944 A.A NLpoly2 1230A.A 5P + N33D + V58A (5A2 + A15S + L18Q + F31I + V58A + A67D + M106V +L149M + V157D) 659 N.A. 5A2 + L18V 945 N.A. NLpoly3 1231 N.A. 5P +I56N + P157H (5A2 + R11N + L18Q + F31I + V58A + A67D + M106V + L149M +V157D) 660 A.A 5A2 + L18V 946 A.A NLpoly3 1232 A.A 5P + I56N + P157H(5A2 + R11N + L18Q + F31I + V58A + A67D + M106V + L149M + V157D) 661N.A. 5A2 + L18W 947 N.A. NLpoly4 1233 N.A. 5P + L46Q + P157H (5A2 +R11N + A15S + F31I + V58A + A67D + M106V + L149M + V157D) 662 A.A 5A2 +L18W 948 A.A NLpoly4 1234 A.A 5P + L46Q + P157H (5A2 + R11N + A15S +F31I + V58A + A67D + M106V + L149M + V157D) 663 N.A. 5A2 + L18Y 949 N.A.NLpoly5 1235 N.A. 5P + I59V (5A2 + R11N + A15S + L18Q + V58A + A67D +M106V + L149M + V157D) 664 A.A 5A2 + L18Y 950 A.A NLpoly5 1236 A.A 5P +I59V (5A2 + R11N + A15S + L18Q + V58A + A67D + M106V + L149M + V157D)665 N.A. 5A2 + F31A 951 N.A. NLpoly6 1237 N.A. 5P + A51T + E74K + (5A2 +R11N + P113L A15S + L18Q + F31I + A67D + M106V + L149M + V157D) 666 A.A5A2 + F31A 952 A.A NLpoly6 1238 A.A 5P + A51T + E74K + (5A2 + R11N +P113L A15S + L18Q + F31I + A67D + M106V + L149M + V157D) 667 N.A. 5A2 +F31C 953 N.A. NLpoly7 1239 N.A. 5P + V36A (5A2 + R11N + A15S + L18Q +F31I + V58A + M106V + L149M + V157D) 668 A.A 5A2 + F31C 954 A.A NLpoly71240 A.A 5P + V36A (5A2 + R11N + A15S + L18Q + F31I + V58A + M106V +L149M + V157D) 669 N.A. 5A2 + F31D 955 N.A. NLpoly8 1241 N.A. 5P + A51T(5A2 + R11N + A15S + L18Q + F31I + V58A + A67D + L149M + V157D) 670 A.A5A2 + F31D 956 A.A NLpoly8 1242 A.A 5P + A51T (5A2 + R11N + A15S +L18Q + F31I + V58A + A67D + L149M + V157D) 671 N.A. 5A2 + F31E 957 N.A.NLpoly9 1243 N.A. 5P + H57R (5A2 + R11N + A15S + L18Q + F31I + V58A +A67D + M106V + V157D) 672 A.A 5A2 + F31E 958 A.A NLpoly9 1244 A.A 5P +H57R (5A2 + R11N + A15S + L18Q + F31I + V58A + A67D + M106V + V157D) 673N.A. 5A2 + F31G 959 N.A. NLpoly10 1245 N.A. 5P + V58A (5A2 + R11N +A15S + L18Q + F31I + V58A + A67D + M106V + L149M) 674 A.A 5A2 + F31G 960A.A NLpoly10 1246 A.A 5P + V58A (5A2 + R11N + A15S + L18Q + F31I +V58A + A67D + M106V + L149M) 675 N.A. 5A2 + F31H 961 N.A. NLpoly11 1247N.A. 5P + E74K (5A2 + A15S + L18Q + M106V + L149M + V157D) 676 A.A 5A2 +F31H 962 A.A NLpoly11 1248 A.A 5P + E74K (5A2 + A15S + L18Q + M106V +L149M + V157D) 677 N.A. 5A2 + F31I 963 N.A. NLpoly12 1249 N.A. 5P + H86Q(5A2 + A15S + L18Q + A67D + M106V + L149M + V157D) 678 A.A 5A2 + F31I964 A.A NLpoly12 1250 A.A 5P + H86Q (5A2 + A15S + L18Q + A67D + M106V +L149M + V157D) 679 N.A. 5A2 + F31K 965 N.A. NLpoly13 1251 N.A. 5P + H93P(5A2 + R11N + A15S + L18Q + M106V + L149M + V157D) 680 A.A 5A2 + F31K966 A.A NLpoly13 1252 A.A 5P + H93P (5A2 + R11N + A15S + L18Q + M106V +L149M + V157D) 681 N.A. 5A2 + F31L 967 N.A. 5P + V 1253 N.A. 5P + I99V682 A.A 5A2 + F31L 968 A.A 5P + V 1254 A.A 5P + I99V 683 N.A. 5A2 + F31M969 N.A. 5P + A 1255 N.A. 5P + K123E 684 A.A 5A2 + F31M 970 A.A 5P + A1256 A.A 5P + K123E 685 N.A. 5A2 + F31N 971 N.A. 5P + VT 1257 N.A. 5P +T128S 686 A.A 5A2 + F31N 972 A.A 5P + VT 1258 A.A 5P + T128S 687 N.A.5A2 + F31P 973 N.A. 5P + VA 1259 N.A. 5P + L142Q + T154N 688 A.A 5A2 +F31P 974 A.A 5P + VA 1260 A.A 5P + L142Q + T154N 689 N.A. 5A2 + F31Q 975N.A. 5P + AT 1261 N.A. 5P + H57Q 690 A.A 5A2 + F31Q 976 A.A 5P + AT 1262A.A 5P + H57Q 691 N.A. 5A2 + F31R 977 N.A. 5P + AA 1263 N.A. 5P + L92M692 A.A 5A2 + F31R 978 A.A 5P + AA 1264 A.A 5P + L92M 693 N.A. 5A2 +F31S 979 N.A. 5P + GG 1265 N.A. 5P + P113L 694 A.A 5A2 + F31S 980 A.A5P + GG 1266 A.A 5P + P113L 695 N.A. 5A2 + F31T 981 N.A. 5P + AA 1267N.A. 5P + G48D 696 A.A 5A2 + F31T 982 A.A 5P + AA 1268 A.A 5P + G48D 697N.A. 5A2 + F31V 983 N.A. 5P + ATG 1269 N.A. 5P − B9 (−147-157) 698 A.A5A2 + F31V 984 A.A 5P + ATG 1270 A.A 5P − B9 (−147-157) 699 N.A. 5A2 +F31W 985 N.A. 5P + VTG 1271 N.A. 5P + L46R + P157S 700 A.A 5A2 + F31W986 A.A 5P + VTG 1272 A.A 5P + L46R + P157S 701 N.A. 5A2 + F31Y 987 N.A.5P + VTA 1273 N.A. 5P + L46H + P157H 702 A.A 5A2 + F31Y 988 A.A 5P + VTA1274 A.A 5P + L46H + P157H 703 N.A. 5A2 + V58A 989 N.A. 5P + GTA 1275N.A. 5P + L46R + H93P 704 A.A 5A2 + V58A 990 A.A 5P + GTA 1276 A.A 5P +L46R + H93P 705 N.A. 5A2 + V58C 991 N.A. 5P + VTGW 1277 N.A. 5P + L46R +H93P + F31L 706 A.A 5A2 + V58C 992 A.A 5P + VTGW 1278 A.A 5P + L46R +H93P + F31L 707 N.A. 5A2 + V58D 993 N.A. 5P + VTGWR 1279 N.A. 5P +L46R + H93P + K75E 708 A.A 5A2 + V58D 994 A.A 5P + VTGWR 1280 A.A 5P +L46R + H93P + K75E 709 N.A. 5A2 + V58E 995 N.A. 5P + VTGWE 1281 N.A.5P + L46R + H93P + I76V 710 A.A 5A2 + V58E 996 A.A 5P + VTGWE 1282 A.A5P + L46R + H93P + I76V 711 N.A. 5A2 + V58F 997 N.A. 5P + VTGWK 1283N.A. 8S (5P + L46R + H93P + P157S + F31L) 712 A.A 5A2 + V58F 998 A.A5P + VTGWK 1284 A.A 8S (5P + L46R + H93P + P157S + F31L) 713 N.A. 5A2 +V58G 999 N.A. 5P + VTGWQ 1285 N.A. 5P + L46R + H93P + P157S + K75E 714A.A 5A2 + V58G 1000 A.A 5P + VTGWQ 1286 A.A 5P + L46R + H93P + P157S +K75E 715 N.A. 5A2 + V58H 1001 N.A. 5P + VTGWH 1287 N.A. 5P + L46R +H93P + P157S + I76V 716 A.A 5A2 + V58H 1002 A.A 5P + VTGWH 1288 A.A 5P +L46R + H93P + P157S + I76V 717 N.A. 5A2 + V58I 1003 N.A. 5P D1 (−157)1289 N.A. 12S (8S + A51T + K75E + I76V + I107L) 718 A.A 5A2 + V58I 1004A.A 5P D1 (−157) 1290 A.A 12S (8S + A51T + K75E + I76V + I107L) 719 N.A.5A2 + V58K 1005 N.A. 5P D2 (−156-157) 1291 N.A. 11S (12-A51T) 720 A.A5A2 + V58K 1006 A.A 5P D2 (−156-157) 1292 A.A 11S (12-A51T) 721 N.A.5A2 + V58L 1007 N.A. 5P D3 (−155-157) 1293 N.A. 12S-K75E 722 A.A 5A2 +V58L 1008 A.A 5P D3 (−155-157) 1294 A.A 12S-K75E 723 N.A. 5A2 + V58M1009 N.A. 5P D4 (−154-157) 1295 N.A. 12S-I76V 724 A.A 5A2 + V58M 1010A.A 5P D4 (−154-157) 1296 A.A 12S-I76V 725 N.A. 5A2 + V58N 1011 N.A. 5PD5 (−153-157) 1297 N.A. 12S-I107L 726 A.A 5A2 + V58N 1012 A.A 5P D5(−153-157) 1298 A.A 12S-I107L

The polypeptides and coding nucleic acid sequences of Table 2 (SEQ IDNOS: 441-1298) all contain N-terminal Met residues (amino acids) or ATGstart codons (nucleic acids). In some embodiments, the polypeptides andcoding nucleic acid sequences of Table 2 are provided without N-terminalMet residues or ATG start codons (SEQ ID NOS: 1299-2156).

In certain embodiments, a polypeptide of one of the amino acid polymersof SEQ ID NOS: 441-2156 is provided. In some embodiments, polypeptidescomprise a single amino acid difference from SEQ ID NO: 440. In someembodiments, polypeptides comprise two or more (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30 . . . 35 . . . 40 . . . 45 . . . 50, or more) aminoacid differences from SEQ ID NO: 440 and/or any of the amino acidpolymers of SEQ ID NOS:441-2156. In some embodiments, polypeptides areprovided comprising the sequence of one of the amino acid polymers ofSEQ ID NOS: 441-2156 with one or more additions, substitutions, and/ordeletions. In some embodiments, a polypeptide or a portion thereofcomprises greater than 70% sequence identity(e.g., >71%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%,or >99%) with one or more of the amino acid polymers of SEQ ID NOS:441-2156.

In certain embodiments, a nucleic acid from Table 2 is provided. In someembodiments, a nucleic acid encoding a polypeptide from Table 2 isprovided. In some embodiments, a nucleic acid of the present inventioncodes for a polypeptide that comprises a single amino acid differencefrom SEQ ID NO: 440 and/or any of the amino acid polymers of SEQ ID NOS:441-2156. In some embodiments, nucleic acids code for a polypeptidecomprising two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 . . .35 . . . 40 . . . 45 . . . 50, or more) amino acid differences from SEQID NO: 440 and/or any of the polypeptides listed in Table 2. In someembodiments, nucleic acids are provided comprising the sequence of oneof the nucleic acid polymers of SEQ ID NOS: 441-2156. In someembodiments, nucleic acids are provided comprising the sequence of oneof the nucleic acid polymers of SEQ ID NOS: 441-2156 with one or moreadditions, substitutions, and/or deletions. In some embodiments, anucleic acid or a portion thereof comprises greater than 70% sequenceidentity(e.g., >71%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%,or >99%) with one or more of the nucleic acid polymers of SEQ ID NOS:441-2156. In some embodiments, a nucleic acid or a portion thereof codesfor an polypeptide comprising greater than 70% sequence identity(e.g., >71%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%,or >99%) with one or more of the amino acid polymers of SEQ ID NOS:441-2156. In some embodiments, nucleic acids are provided that code forone of the polypeptides of SEQ ID NOS: 441-2156. In some embodiments,nucleic acids are provided that code for one of the polypeptides of SEQID NOS: 441-2156 with one or more additions, substitutions, and/ordeletions.

In some embodiments, a non-luminescent peptide or polypeptide and/or aninteraction element, comprises a synthetic peptide, peptide containingone or more non-natural amino acids, peptide mimetic, conjugatedsynthetic peptide (e.g., conjugated to a functional group (e.g.,fluorophore, luminescent substrate, etc.)).

The present invention provides compositions and methods that are usefulin a variety of fields including basic research, medical research,molecular diagnostics, etc. Although the reagents and assays describedherein are not limited to any particular applications, and any usefulapplication should be viewed as being within the scope of the presentinvention, the following are exemplary assays, kits, fields,experimental set-ups, etc. that make use of the presently claimedinvention.

Typical applications that make use of embodiments of the presentinvention involve the monitoring/detection of protein dimerization(e.g., heterodimers, homodimers), protein-protein interactions,protein-RNA interactions, protein-DNA interactions, nucleic acidhybridization, protein-small molecule interactions, or any othercombinations of molecular entities. A first entity of interest isattached to a first member of a non-luminescent pair and the secondentity of interest is attached to the second member of a non-luminescentpair. If a detectable signal is produced under the particular assayconditions, then interaction of the first and second entities areinferred. Such assays are useful for monitoring molecular interactionsunder any suitable conditions (e.g., in vitro, in vivo, in situ, wholeanimal, etc.), and find use in, for example, drug discovery, elucidatingmolecular pathways, studying equilibrium or kinetic aspects of complexassembly, high throughput screening, proximity sensor, etc.

In some embodiments, a non-luminescent pair of known characteristics(e.g., spectral characteristics, mutual affinity of pair) is used toelucidate the affinity of, or understand the interaction of, aninteraction pair of interest. In other embodiments, a well-characterizedinteraction pair is used to determine the characteristics (e.g.,spectral characteristics, mutual affinity of pair) of a non-luminescentpair.

Embodiments described herein may find use in drug screening and/or drugdevelopment. For example, the interaction of a small molecule drug or anentire library of small molecules with a target protein of interest(e.g., therapeutic target) is monitored under one or more relevantconditions (e.g., physiological conditions, disease conditions, etc.).In other embodiments, the ability of a small molecule drug or an entirelibrary of small molecules to enhance or inhibit the interactionsbetween two entities (e.g., receptor and ligand, protein-protein, etc.)is assayed. In some embodiments, drug screening applications are carriedout in a high through-put format to allow for the detection of thebinding of tens of thousands of different molecules to a target, or totest the effect of those molecules on the binding of other entities.

In some embodiments, the present invention provides the detection ofmolecular interactions in living organisms (e.g., bacteria, yeast,eukaryotes, mammals, primates, human, etc.) and/or cells. In someembodiments, fusion proteins comprising signal and interaction (target)polypeptides are co-expressed in the cell or whole organism, and signalis detected and correlated to the formation of the interaction complex.In some embodiments, cells are transiently and/or stably transformed ortransfected with vector(s) coding for non-luminescent element(s),interaction element(s), fusion proteins (e.g., comprising a signal andinteraction element), etc. In some embodiments, transgenic organisms aregenerated that code for the necessary fusion proteins for carrying outthe assays described herein. In other embodiments, vectors are injectedinto whole organisms. In some embodiments, a transgenic animal or cell(e.g., expressing a fusion protein) is used to monitor thebiodistribution of a small molecule or a biologic tethered (e.g.,conjugated or genetically fused) to NLpeptide sequence that would form acomplex in the subcellular compartments and/or tissues where itconcentrates.

In some embodiments, a peptide (e.g., non-luminescent peptide) portionof a luminescent complex is employed as a protein tag (e.g., withincells). In such embodiments, a polypeptide (e.g., non-luminescentpolypeptide) portion of a luminescent complex (e.g., capable of forminga luminescent complex with the non-luminescent peptide) is applied tocells (e.g., as part of a reagent) to detect/quantify the presence ofproteins tagged with the non-luminescent peptide. For example, a proteinof interest is fused to a high affinity NLpep (e.g., NLpep86). The NLpepis then transfected into cells of interest, a reagent containingNanoGlo+NLpoly 11S is then added to cells+media, and luminescence isdetected. This assay scheme is demonstrated in FIG. 175. In someembodiments, the small size of the peptide is useful for proteintagging. In some embodiments, non-luminescent polypeptides used in sucha system are stable enough to exist in a suitable buffer for extendedperiods of time (e.g., in the presence of the furimazine substrate). Incertain embodiments, the non-luminescent polypeptide has minimaldetectable luminescence in the absence of the complementing peptide(e.g., even in the presence of furimazine substrate). In someembodiments, optimized buffer conditions are utilized to meet criterianecessary for protein tagging. High affinity spontaneously polypeptidesand peptides are useful in such systems, and have utility in, forexample, immunoassays, detection of virus particles, the study ofprotein dynamics in living cells, etc. In some embodiments, such asystem provides an extremely small protein tag (e.g., 11 amino acids)providing high sensitivity detection, stability (e.g., particularlyunder denaturing conditions), and/or a broad dynamic range.

The compositions and methods provided herein, as well as any techniquesor technologies based thereon find use in a variety of applications andfields, a non-limiting list of example applications follows:

-   -   Antibody-free Western Blot: For example, a protein of interest        is fused to a non-luminescent peptide (e.g., by genetic        engineering) and expressed by any suitable means. The proteins        separated (e.g., by PAGE) and transferred to a membrane. The        membrane is then washed with complimentary non-luminescent        polypeptide (e.g. allowing a luminescent complex to form), and        placed on imager (e.g., utilizing a CCD camera) with Furimazine        (PBI-3939) atop the membrane, and the protein of interest is        detected (e.g., via the luminescence of the luminescent        complex).    -   “LucCytochemistry”: For example, a protein of interest is        expressed fused to a non-luminescent peptide or polypeptide and        then detected with a complimentary non-luminescent polypeptide        or peptide in a fashion analogous to immunocytochemistry.    -   Protein localization assay: For example, a localization signal        is added to a non-luminescent polypeptide or polypeptide (e.g.,        via genetic engineering) and expressed in cells (e.g., a nuclear        localization signal added would result in expression of the        non-luminescent polypeptide in the nucleus). A complimentary        non-luminescent peptide or polypeptide is fused to a protein of        interest (e.g., via genetic engineering) and expressed in cells        with the non-luminescent polypeptide or peptide. Luminescence is        produced if the protein of interest localizes in the same        subcellular compartment (e.g., the nucleus) as the        signal-localized non-luminescent polypeptide.    -   Protein Stability Assay: For example, a protein of interest is        fused to a non-luminescent peptide or polypeptide (e.g., via        genetic engineering) and incubated under one or more conditions        of interest. A complimentary non-luminescent polypeptide or        peptide is added (e.g., at various time points), and        luminescence is used to quantify the amount of protein of        interest (e.g., a proxy for stability).    -   Protein Detection/Quantification: For example, a protein of        interest fused to a non-luminescent peptide or polypeptide        (e.g., via genetic engineering) and expressed and/or manipulated        by any method. The complimentary non-luminescent polypeptide or        peptide is then added to detect and/or quantify the protein of        interest.    -   Protein Purification: For example, a protein of interest is        fused to a non-luminescent peptide or polypeptide (e.g., via        genetic engineering) and expressed by any method. The mixture of        proteins is passed through an immobilized complimentary        non-luminescent polypeptide or peptide (e.g., on beads, on a        column, on a chip, etc.), washed with suitable buffer and eluted        (e.g., with a buffer of high ionic strength or low pH). A mutant        form of the non-luminescent peptide or polypeptide that does not        activate the luminescence of the complimentary non-luminescent        peptide or polypeptide may be used to elute the protein of        interest.    -   Pull-down: For example, an immobilized, complimentary,        non-luminescent polypeptide is used to isolate a protein of        interest (and interacting proteins) that is fused to a        non-luminescent peptide (e.g., via genetic engineering).    -   G-Coupled Protein Receptor (GPCR) Internalization Assay: For        example, a non-luminescent peptide or polypeptide is fused to a        GPCR of interest (e.g., via genetic engineering) and expressed        on the surface of cells. A complimentary non-luminescent        polypeptide or peptide is added to the media of the cells and        used to detect the GPCR on cell surface. A ligand is added to        stimulate the internalization of the GPCR, and a decrease in        luminescence is observed.    -   Membrane Integrity Assay for Cell Viability: For example, when        the cell membrane of a cell expressing a non-luminescent        polypeptide become compromised, a non-luminescent peptide enters        the cell (e.g., a peptide that otherwise can't cross the cell        membrane), thereby forming a luminescent complex, and generating        luminescence.    -   5-Hydroxymethyl Cytosine Detection: For example, a cysteine is        added to a non-luminescent peptide and incubated with DNA and a        methyltransferase. The methyltransferase catalyzes the addition        of the thiol (cysteine) only onto cytosine residues that are        5-hydroxymethylated. Unincorporated peptide is then separated        from the DNA (using any method possible), and a non-luminescent        polypeptide is added to detect the peptide conjugated to the        DNA.    -   Formyl Cytosine Detection: For example, similar to the        5-hydroxymethyl cytosine detection above, this detection method        uses chemistry with specific reactivity for formyl cytosine.    -   Viral Incorporation: Nucleic acid coding for a non-luminescent        peptide or polypeptide is incorporated into a viral genome, and        the complementary non-luminescent polypeptide or peptide is        constitutively expressed in the target cells. Upon infection of        the target cells and expression of the non-luminescent peptide,        the bioluminescent complex forms and a signal is detected (e.g.,        in the presence of substrate).    -   Chemical Labeling of Proteins: A non-luminescent peptide is        fused or tethered to a reactive group (e.g., biotin,        succinimidyl ester, maleimide, etc.). A protein of interest        (e.g., antibody) is tagged with the non-luminescent peptide        through binding of the reactive group to the protein of        interest. Because the peptide is small, it does not affect the        functionality of the protein of interest. Complimentary        non-luminescent polypeptide is added to the system, and a        luminescent complex is produced upon binding to the polypeptide        to the peptide.    -   Protease Assay: For example, a peptide sequence that is        recognized by a protease of interest can be joined to NLPep in        such a way that prevents bioluminescence upon exposure to        NLPoly. Ways to do this include attaching a luminescence        quencher to the protease recognition sequence or binding the        protease recognition sequence to NLPep in such a way that        complementation is hindered. Upon activity of the protease to        cleave the recognition sequence, the ability of NLPoly to        complement to NLPep and emit luminescence is restored, and thus        the system is a sensitive protease assay.    -   RNA detection.    -   Biomolecule Linker characterization: For example, a linker        attached to a biomolecule such as an antibody can be evaluated        for its stability under a set of conditions through attaching        NLPep to the molecule via the linker of interest. Over time, the        production of free NLPep through linker degradation can be        monitored by addition of NLPoly and furimazine and        quantification of bioluminescence produced.    -   Mutation assay: For example, a point mutation, a frameshift        mutation, etc. introduced in vitro or in vivo results in either        a gain of signal or loss of signal from a complementation pair.        Such an assay could be used, for example, to test compounds for        mutagenicity.    -   Target engagement for peptide inhibitors: Use of low affinity        NLpep-conjugated peptides (expressed in cells) to monitor target        engagement of peptide-based inhibitors. NLpoly is tethered to        the target of interest. Engagement results in loss of signal        from luminescent complex.    -   Gain of signal Protease biosensors: A protease cleavage site is        expressed between NLpoly and a dark peptide NLpep (low        affinity). Cleavage releases dark peptide allowing for high        affinity NLpep to complement NLpoly.    -   Gain of function protease assay: The sequence of an NLpep is        engineered proximal to a cleavage site of a full length        substrate for a protease (e.g., caspase, ADAM, etc). The peptide        remains sterically inaccessible as long as the substrate remains        intact and the peptide is “buried”. Both the genetically        engineered protease substrate and a NLpoly (e.g., NLpoly 11S)        are co-transfected into a target cell line. Luciferase activity        is induced upon induction of protease activity which leads to        the cleavage of the substrate and exposure of the activator        peptide on the N- or C-terminus of one of the fragments. This        principle is expandable to detect conformational changes and/or        protein modifications as well.    -   Intracellular analyte quantification using recombinant        intrabodies: Antibody fragments expressed within cells as NLpoly        or NLpep fusion. Complementary subunit is genetically fused to        an analyte of interest. When analyte is present, antibody binds        and luminescent complex is formed. The application is expandable        to intracellular PTM (e.g. phosphorylation) biosensors, in which        the intrabody only binds to the analyte when it has been        phosphorylated (or otherwise bound by the modification-specific        Ab).        The above applications of the compositions and methods of the        present invention are not limiting and may be modified in any        suitable manner while still being within the scope of the        present invention.

The present invention also provides methods for the design and/oroptimization of non-luminescent pairs/groups and the bioluminescentcomplexes that form therefrom. Any suitable method for the design ofnon-luminescent pairs/groups that are consistent with embodimentsdescribed herein, and/or panels thereof, is within the scope of thepresent invention.

In certain embodiments, non-luminescent pairs/groups are designed denovo to lack luminescence individually and exhibit luminescence uponassociation. In such embodiments, the strength of the interactionbetween the non-luminescent elements is insufficient to produce abioluminescent signal in the absence of interaction elements tofacilitate formation of the bioluminescent complex.

In other embodiments, non-luminescent elements and/or non-luminescentpairs are rationally designed, for example, using a bioluminescentprotein (e.g., SEQ ID NO: 2157) as a starting point. For example, suchmethods may comprise: (a) aligning the sequences of three or morerelated proteins; (b) determining a consensus sequence for the relatedproteins; (c) providing first and second fragments of a bioluminescentprotein that is related to the ones from which the consensus sequencewas determined, wherein the fragments are individually substantiallynon-luminescent but exhibit luminescence upon interaction of thefragments; (d) mutating the first and second fragments at one or morepositions each (e.g., in vitro, in silico, etc.), wherein said mutationsalter the sequences of the fragments to be more similar to acorresponding portion of the consensus sequence, wherein the mutatingresults in a non-luminescent pair that are not fragments of apreexisting protein, and (e) testing the non-luminescent pair for theabsence of luminescence when unassociated and luminescence uponassociation of the non-luminescent pair. In other embodiments, first andsecond fragments of one of the proteins used in determining theconsensus sequence are provided, mutated, and tested.

In some embodiments, a peptide of a luminescent pair is a ‘darkpeptide,’ or one that binds to its complement (e.g., NLpoly) (e.g., withlow or high affinity) but produces minimal or no luminescence (See FIGS.180-182). In some embodiments, a high affinity dark peptide finds use ininverse complementation, or gain of signal assays for measuringinhibitors. In some embodiments, a low affinity dark peptide is used tobring down background of NLpoly11S in a reagent for the detection of ahigh affinity peptide tag (e.g. NLpep86). Exemplary dark peptides areprovided in FIG. 180.

In some embodiments, a peptide of a luminescent pair is a ‘quencherpeptide,’ or one that contains a quencher moiety (e.g., DAB), and thequencher absorbs the light/energy produced by both a NLpoly in isolation(e.g., the signal produced independent of a complementing NLpep) and aNLpoly-NLpep complex (e.g., the signal produced as a result of complexformation). Exemplary dark quencher peptides would have a suitableabsorption spectrum and include DAB-161 (DAB-GWRLFKK (SEQ ID NO: 2370)),DAB-162 (DAB-GWALFKK (SEQ ID NO: 2351)), DAB-163 (DAB-VTGWALFEEIL (SEQID NO: 2372)), DAB-164 (DAB-VTGYALFQEIL (SEQ ID NO: 2573)), DAB-165(DAB-VTGYALFEQIL (SEQ ID NO: 2574), and DAB-166 (DAB-VTGYALFEEIL (SEQ IDNO: 2575); wherein DAB=Dabcyl (475 nm quencher)+dPEG4 spacer.

In some embodiments, the above methods are not limited to the designand/or optimization of non-luminescent pairs. The same steps areperformed to produce pairs of elements that lack a given functionality(e.g., enzymatic activity) individually, but display such functionalitywhen associated. In any of these cases, the strength of the interactionbetween the non-luminescent pair elements may be altered via mutationsto ensure that it is insufficient to produce functionality in theabsence of interaction elements that facilitate formation of thebioluminescent complex.

EXPERIMENTAL Example 1 Generation of Peptides

Peptide constructs were generated by one of three methods: annealing5′-phosphorylated oligonucleotides followed by ligation topF4Ag-Barnase-HALOTAG vector (Promega Corporation; cut with SgfI andXhoI) or pFN18A (Promega Corporation; cut with SgfI and XbaI), sitedirected mutagenesis using Quik Change Lightning Multi kit from Agilentor outsourcing the cloning to Gene Dynamics.

Example 2 Peptide Preparation

The peptides generated in Example 1 were prepared for analysis byinoculating a single colony of KRX E. coli cells (Promega Corporation)transformed with a plasmid encoding a peptide into 2-5 ml of LB cultureand grown at 37° C. overnight. The overnight cultures (10 ml) were thendiluted into 1 L of LB and grown at 37° C. for 3 hours. The cultureswere then induced by adding 10 ml 20% rhamnose to the 1 L culture andinduced at 25° C. for 18 hours.

After induction, 800 ml of each culture was spun at 5000×g at 4° C. for30 minutes. The pellet generated was then resuspended in 80 ml PeptideLysis Buffer (25 mM HEPES pH 7.4, 0.1× Passive Lysis Buffer (PromegaCorporation), 1 ml/ml lysozyme and 0.03 U/μl RQ1 DNase (PromegaCorporation)) and incubated at room temperature for 15 minutes. Thelysed cells were then frozen on dry ice for 15 minutes and then thawedin a room temperature bath for 15 minutes. The cells were then spun at3500×g at 4° C. for 30 minutes. The supernatants were aliquoted into 10ml samples with one aliquot of 50 μl placed into a 1.5 ml tube.

To the 50 μl samples, 450 μl H₂O and 167 μl 4×SDS Loading Dye wereadded, and the samples incubated at 95° C. for 5 minutes. After heating,5 μl of each sample was loaded (in triplicate) onto an SDS-PAGE gel, andthe gel run and stained according to the manufacturer's protocol. Thegel was then scanned on a Typhoon Scanner (excitation 532 nm, emission580 nm, PMT sensitivity 400V). The resulting bands were quantified usingthe ImageQuant (5.2) software. Each of the three replicate intensitieswas averaged, and the average intensity of NLpep53-HT was defined at 12×concentration. The concentrations of all other peptides were relative toPep53-HT.

Example 3 Peptide Analysis

All of the peptides generated in Examples 1-2 contained single mutationsto the peptide sequence: GVTGWRLCKRISA (SEQ ID NO: 236). All of thepeptides were fused to a HALOTAG protein (Promega Corporation). Peptidesidentified as “HT-NLpep” indicate that the peptide is located at theC-terminus of the HALOTAG protein. In this case, the gene encoding thepeptide includes a stop codon, but does not include a methionine toinitiate translation. Peptides identified as “NLpep-HT” indicate thatthe peptide is at the N-terminus of the HALOTAG protein. In this case,the peptide does include a methionine to initiate translation, but doesnot include a stop codon.

To determine the ability of the peptides to activate luminescence,individual colonies of KRX E. coli cells (Promega Corporation) wastransformed with a plasmid encoding a peptide from Example 1, inoculatedin 200 μl of minimal medium (1×M9 salts, 0.1 mM CaCl₂), 2 mM MgSO₄, 1 mMThiamine HCl, 1% gelatin, 0.2% glycerol, and 100 ul/ml Ampicillin) andgrown at 37° C. overnight. In addition to the peptides, a culture of KRXE. coli cells expressing a wild-type (WT) fragment of residues 1-156 ofthe NanoLuc was grown. All peptides and the WT fragment were inoculatedinto at least 3 separate cultures.

After the first overnight growth, 10 μl of culture was diluted into 190μl fresh minimal medium and again grown at 37° C. overnight.

After the second overnight growth, 10 μl of the culture was diluted into190 μl of auto-induction medium (minimal medium+5% glucose and 2%rhamnose). The cultures were then inducted at 25° C. for approximately18 hours.

After induction, the small peptide mutant cultures were assayed foractivity. The cultures containing the WT 1-156 fragment were pooled,mixed with 10 ml of 2× Lysis Buffer (50 mM HEPES pH 7.4, 0.3× PassiveLysis Buffer, and 1 mg/ml lysozyme) and incubated at room temperaturefor 10 minutes. 30 μl of the lysed WT 1-156 culture was then aliquotedinto wells of a white, round bottom 96-well assay plate (Costar 3355).To wells of the assay plate, 20 μl of a peptide culture was added, andthe plate incubated at room temperature for 10 minutes. Afterincubation, 50 μl NANOGLO Luciferase Assay Reagent (Promega Corporation)was added, and the samples incubated at room temperature for 10 minutes.Luminescence was measured on a GLOMAX luminometer with 0.5 sintegrations.

The results (See Table 3 and FIG. 1) demonstrate various mutations inthe peptide (relative to SEQ ID NO: 1) that altered (e.g., increased,decreased) the luminescence following complementation with the wild-typenon-luminescent polypeptide. The increased luminescence is thought tostem from one (or a combination) of five main factors, any of which arebeneficial: affinity between the non-luminescent peptide andnon-luminescent polypeptide, expression of the peptide, intracellularsolubility, intracellular stability, and bioluminescent activity. Thepresent invention though is not limited to any particular mechanism ofaction and an understanding of the mechanism of action is not necessaryto practice the present invention.

TABLE 3 Pep-HT st. Mutation HT-NLPep NLpep-HT HT-Pep st. dev. dev. G157D0.1137 0.5493 N.D. N.D. G157N 0.6415 3.3074 0.2512 1.4828 G157S 1.99371.7156 0.8554 1.0563 G157E 0.1959 1.4461 0.0811 0.3221 G157H 0.93800.5733 0.4366 0.2277 G157C N.D. 0.0468 N.D. 0.0081 G157P N.D. 0.0543N.D. 0.0106 V158I 0.6075 1.6010 0.3283 0.6264 V158A 0.1348 0.1438 0.05610.0447 V158K 0.0770 0.1923 0.0323 0.0521 V158Q 0.0445 0.0397 0.01880.0160 V158S 0.0487 0.0838 0.0189 0.0251 T159V 0.5658 0.0455 0.22930.0005 T159K 0.0490 0.0307 0.0120 0.0103 T159Q 0.3979 0.0310 0.10630.0091 W161T 0.0028 0.0100 0.0007 0.0049 W161K 0.0002 0.0008 9.7E−060.0001 W161V 0.0086 0.0050 0.0062 0.0016 W161F N.D. 0.0717 N.D. 0.0049W161Y N.D. 0.2154 N.D. 0.0103 W161E N.D. 0.0012 N.D. 0.0002 L163I N.D.0.2923 N.D. 0.1198 L163V 0.1727 0.1190 0.0257 0.0288 L163T 0.0259 0.02620.0077 0.0122 L163Y 0.0512 0.1959 0.0126 0.1043 L163K 0.0885 0.07860.0130 0.0244 C164N 0.0874 0.1081 0.0097 0.0160 C164T 0.0116 0.00840.0029 0.0013 C164F N.D. 13.3131  N.D. 3.6429 C164Y N.D. 1.0092 N.D.0.2592 C164S N.D. 0.0202 N.D. 0.0029 C164H N.D. 0.7597 N.D. 0.2149 C164MN.D. 3.2618 N.D. 1.1763 C164A N.D. 0.0858 N.D. 0.0196 C164Q N.D. 0.0211N.D. 0.0044 C164L N.D. 1.0170 N.D. 0.2464 C164K N.D. 0.0005 N.D. 0.0001R166K 1.0910 1.2069 0.2266 0.5913 R166N 0.1033 0.1182 0.0289 0.0542I167V 0.8770 1.0824 0.1113 0.2642 I167Q 0.0178 0.1172 0.0252 0.0150I167E 0.2771 0.2445 0.0358 0.0456 I167R 0.0464 0.0469 0.0027 0.0084I167F 0.2832 0.1793 0.0159 0.0683 A169N 0.9115 1.7775 0.1114 0.5901A169T 0.9448 1.3720 0.0930 0.6021 A169R 0.9851 0.5014 0.2205 0.1895A169L 1.1127 0.9047 0.1906 0.2481 A169E 0.8457 0.7889 0.1445 0.0819

Example 4 Generation of Non-Luminescent Polypeptides

Using pF4Ag-NanoLuc1-156 (WT 1-156) as a template, error-prone PCR(epPCR) was performed using the Diversify PCR Random Mutagenesis Kitfrom Clontech. The resulting PCR product was digested with SgfI and XbaIand ligated to pF4Ag-Barnase (Promega Corporation), a version of thecommercially-available pF4A vector (Promega) which contains T7 and CMVpromoters and was modified to contain an E. coli ribosome-binding site.Following transformation into KRX E. coli cells (Promega Corporation) byheat shock at 42° C., individual colonies were used to inoculate 200 μlcultures in clear, flat bottom 96-well plates (Costar 3370).

Example 5 Non-Luminescent Polypeptide Analysis

To determine the luminescence of the non-luminescent polypeptide mutantsgenerated in Example 4, individual colonies of the KRX E. coli cells(Promega Corporation) transformed with a plasmid containing one of thenon-luminescent polypeptide mutants from Example 4 was grown accordingto the procedure used in Example 3. The bacterial cultures were alsoinduced according to the procedure used in Example 3.

To assay each non-luminescent polypeptide mutant induced culture, 30 μlof assay lysis buffer (25 mM HEPES pH 7.4, 0.3× Passive Lysis Buffer(Promega Corporation)), 0.006 U/μl RQ1 DNase (Promega Corporation) and1× Peptide Solution (the relative concentration of the peptides weredetermined as explained in Example 2; from the relative concentrationdetermined, the peptides were diluted to 1× in the lysis buffer)containing either the peptide fragment GVTGWRLCKRISA (SEQ ID NO: 18) orGVTGWRLFKRISA (SEQ ID NO: 106) were aliquoted into wells of a 96-wellassay plate (Costar 3355). To the wells of the assay plate, 20 μl of aninduced non-luminescent polypeptide mutant culture was added, and theplate incubated at room temperature for 10 minutes. After incubation, 50μl of NANOGLO Luciferase Assay Reagent (Promega Corporation) was added,and the samples incubated at room temperature for 10 minutes.Luminescence was measured on a GLOMAX luminometer with 0.5 sintegrations.

The results (Table 4 and FIG. 2) demonstrate numerous point mutationsthat improve the luminescence of the non-luminescent polypeptide uponcomplementation with two different peptides. Similar to the mutations inthe peptide, these mutations in the non-luminescent polypeptide may stemfrom various factors, all of which are beneficial to the system as awhole.

TABLE 4  Mutation V157D F311 L18Q R11N GVTGWRLCKRISA 4.98 4.1  3.81 3.37st dev 0.48 0.37 0.29 0.67 GVTGWRLFKRISA 3.02 2.83 2.99 2.09 st dev 0.770.61 0.82 0.03 Mutation Q32R M106V M106I G67S GVTGWRLCKRISA 1.52 1.3 1.27 1.22 st dev 0.2  0.22 0.04 0.26 GVTGWRLFKRISA 1.04 1.4  1.31 1.29st dev 0.19 0.25 0.35 0.22 Mutation F31L L149M N33K I59T GVTGWRLCKRISA3.13 2.89 2.15 1.07 st dev 0.26 0.39 0.2  0.07 GVTGWRLFKRISA 2.86 2.161.76 1.35 st dev 0.7  0.26 0.08 0.37 Mutation I56N T13I F31V N33RGVTGWRLCKRISA 0.44 2.18 2.12 2.1  st dev 0.05 0.75 0.09 0.18GVTGWRLFKRISA 1.81 1.44 2.12 1.56 st dev 0.35 0.34 0.46 0.16 MutationV27M Q20K V58A K75E GVTGWRLCKRISA 1.99 4.43 1.88 2.08 st dev 0.09 0.840.6  0.47 GVTGWRLFKRISA 1.7  2.33 1.07 2.05 st dev 0.11 0.38 0.26 0.37Mutation G15S G67D R112N N156D GVTGWRLCKRISA 1.98 1.78 1.61 1.57 st dev0.99 0.11 0.2  0.21 GVTGWRLFKRISA 2.34 1.57 1.45 1.21 st dev 0.82 0.170.47 0.26 Mutation D108N N144T N156S GVTGWRLCKRISA 2.08 3.69 1.04 st dev0.6  1.12 0.29 GVTGWRLFKRISA 1.88 2.26 1.4  st dev 0.38 0.51 0.28 *Unitsin Table 4 are RLU(mutant)/RLU(VT)

Example 6 Glycine to Alanine Substitutions in Non-LuminescentPolypeptide

The following example identified glycine residues within thenon-luminescent polypeptide that can be substituted to alanine toprovide an improved (e.g., greater luminescent signal) non-luminescentpolypeptide. The substitutions were made singly (See FIG. 3), or incomposites (FIG. 2). Non-luminescent polypeptides containing glycine toalanine substitutions were generated as described in Example 1.

Each single mutant colony was inoculated in 200 μl Minimal Media (1×M9salts, 0.1 mM CaCl₂), 2 mM MgSO₄, 1 mM Thiamine HCl, 1% gelatin, 0.2%glycerol and 1×ampicillin) and incubated with shaking at 37° C. for 20hours. 10 μl of the culture was then added to 190 μl of fresh MinimalMedia and incubated again with shaking at 37° C. for 20 hours. 10 μl ofthe second culture was then added to 190 μl Auto-Induction Media(Minimal Media+5% glucose+2% rhamnose) and incubated with shaking at 25°C. for 18 hours to allow expression of the non-luminescent polypeptide.

To assay each mutant culture, 30 μl of assay lysis buffer (50 mM HEPESpH 7.5, 0.3× Passive Lysis Buffer (Promega Corporation)) and 0.006 U/μlRQ1 DNase (Promega Corporation)) containing non-luminescent peptide(1:10 dilution of NLpep9-HT (NLpep9 is SEQ ID NO: 17 and 18; HT isHaloTag E. coli clarified lysate) was added. The samples were shaken atroom temperature for 10 minutes, and then 50 μl NANOGLO Luciferase AssayReagent (Promega Corporation) was added. The samples were incubated atroom temperature for 10 minutes, and luminescence was measured on aGLOMAX luminometer with 0.5 s integrations.

To generate the NLpep9-HT E. coli clarified lysate, 5 ml LB wasinoculated with a single E. coli colony of NLpep9-HT and incubated at37° C. overnight. 500 μl of the overnight culture was then diluted in 50mls LB and incubated at 37° C. for 3 hours. 500 μl of 20% rhamnose wasadded and incubated at 25° C. for 18 hours. The expression culture wascentrifuged at 3000×g for 30 minutes, and the cell pellet resuspended in5 ml peptide lysis buffer (25 mM HEPES, pH 7.5, 0.1× Passive LysisBuffer, 1 mg/ml lysozyme, and 0.3 U/μl RQ1 DNase) and incubated at roomtemperature for 10 minutes. The lysed sample was placed on dry ice for15 minutes, thawed in a room temperature water bath and centrifuged at3500×g for 30 minutes. The supernatant was the clarified lysate.

FIGS. 3 and 4 demonstrate the effects of the mutations on luminescence.

Example 7 Mutations in Non-Luminescent Peptide

In the following example, mutations were made in the non-luminescentpeptide based on alignment to other fatty acid binding proteins (FABPs)and were chosen based on high probability (frequency in FABPs) toidentify a mutation that retains/improves activity (such as NLpep2, 4,and 5) or establish that a mutation is not likely to be tolerated atthat position (such as NLpep3). NLpep1-5 contain single mutations (SeeTable 1), and NLpep6-9 are composite sets of the mutations in NLpep2, 4,and 5 (See Table 1). Mutants were generated as described in Example 1.

Each mutant colony was inoculated in 200 μl Minimal Media and incubatedwith shaking at 37° C. for 20 hours. 10 μl of the culture was then addedto 190 μl of fresh Minimal Media and incubated again with shaking at 37°C. for 20 hours. 10 μl of the second culture was then added to 190 μlAuto-Induction Media and incubated with shaking at 25° C. for 18 hoursto allow expression of the non-luminescent peptide mutant.

To assay each mutant culture, 30 μl of assay lysis buffer (50 mM HEPESpH 7.5, 0.3× Passive Lysis Buffer (Promega Corporation)) and 0.006 U/μlRQ1 DNase (Promega Corporation)) containing non-luminescent polypeptide(1:10 dilution of wild-type non-luminescent polypeptide E. coliclarified lysate) was added. The samples were shaken at room temperaturefor 10 minutes, and then 50 μl NANOGLO Luciferase Assay Reagent (PromegaCorporation) added. The samples were incubated at room temperature for10 minutes, and luminescence was measured on a GLOMAX luminometer with0.5 s integrations.

FIG. 1 shows the luminescence (RLUs) detected in each non-luminescentpeptide mutant. The results demonstrate various positions that are ableto tolerate a mutation without substantial loss in luminescence, as wellas a few specific mutations that improve luminescence.

Example 8 Effect of Orientation of Fusion Tag on Luminescence

In the following example, luminescence generated by non-luminescentpeptides with N- or C-terminus HaloTag protein was compared.

Single colony of each peptide-HT fusion was grown according to theprocedure used in Example 7. The bacterial cultures were also inducedaccording to the procedure used in Example 7. Luminescence was assayedand detected according to the procedure used in Example 7. FIGS. 6 and 7demonstrate the luminescence (RLUs) detected in each peptide-HT fusion.The results demonstrate combinations of mutations that produce similarluminescence as NLpep1.

Example 9 Effect of Multiple Freeze-Thaw Cycles on Non-LuminescentPeptides

1 ml of NLpep9-HT was frozen on dry ice for 5 minutes and then thawed ina room temperature water bath for 5 minutes. 60 μl was then removed forassaying. The freeze-thaw procedure was then repeated another 10 times.After each freeze-thaw cycle, 60 μl of sample was removed for assaying.

To assay, 20 μl of each freeze-thaw sample was mixed with 30 μl of SEQID NO:2 and incubated at room temperature for 10 minutes. 50 μl ofNANOGLO Luciferase Assay Reagent was added, and the samples incubated atroom temperature for 10 minutes. Luminescence was measured on a GLOMAXluminometer with 0.5 s integrations. The results are depicted in FIG. 8and demonstrate that NLpep can be subjected to multiple freeze-thawcycles without a loss in activity (luminescence).

Example 10 Distinction of Mutations in Non-Luminescent Peptides

In the following example, TMR gel analysis was used to normalize theconcentration of the non-luminescent peptide mutants to distinguishmutations that alter the expression from those that alter luminescence(e.g., altered luminescence may stem from altered binding affinity).

5 ml of LB was inoculated with a single mutant peptide colony andincubated with shaking at 37° C. for 20 hours. 50 μl of the overnightculture was diluted into 5 ml of fresh LB and incubated with shaking at37° C. for 3 hours. 50 μl of 20% rhamnose was then added and incubatedwith shaking at 25° C. for 18 hours.

For TMR gel analysis, 79 μl of each induced culture was mixed with 10 μl10× Fast Break Lysis Buffer (Promega Corporation), 10 μl of a 1:100dilution of HALOTAG TMR ligand (Promega Corporation) non-luminescentpolypeptide and 10 μl of RQ1 DNase and incubated at room temperature for10 minutes. 33.3 μl of 4×SDS-loading buffer was added, and the samplesincubated at 95° C. for 5 minutes. 15 μl of each sample was loaded ontoan SDS gel and run according to the manufacturer's directions. The gelwas then scanned on a Typhoon.

Each culture was diluted based on the TMR-gel intensity to normalizeconcentrations. 20 μl of each diluted culture was then mixed with 30 μlassay lysis buffer containing non-luminescent polypeptide (1:10 dilutionof SEQ ID NO: 2 E. coli clarified lysate) and incubated with shaking atroom temperature for 10 minutes. 50 μl of NANOGLO Luciferase AssayReagent was added, and the samples incubated at room temperature for 10minutes. Luminescence was measured on a GLOMAX luminometer with 0.5 sintegrations (SEE FIG. 9).

Example 11 Site Saturation in Non-Luminescent Polypeptide

In the following example, positions 11, 15, 18, 31, 58, 67, 106, 149,and 157 were identified as sites of interest from screening the libraryof random mutations in wild-type non-luminescent polypeptide. All 20amino acids at these positions (built on 5A2 non-luminescent mutantgenerated in Example 6 (SEQ ID NOS: 539 and 540) to validate with othermutations in the 5A2 mutant) were compared to determine the optimalamino acid at that position. Mutant non-luminescent polypeptides weregenerated as previously described in Example 1. Single colony of eachnon-luminescent polypeptide mutant was grown according to the procedureused in Example 6. The bacterial cultures were also induced according tothe procedure used in Example 6. Luminescence was assayed and detectedaccording to the procedure used in Example 6 expect NLpep53 E. coliclarified lysate was used at 1:11.85 dilution.

FIGS. 10-18 demonstrate the effect of the mutations on the ability toproduce luminescence with and without NLpep.

Example 12 Comparison of Cysteine Vs. Proline as First Amino Acid inNon-Luminescent Peptide

In the following example, a comparison of using cysteine or proline asfirst amino acid (after necessary methionine) in the non-luminescentpeptide was performed. The mutant non-luminescent peptides weregenerated as previously described in Example 1. Single colony of eachnon-luminescent polypeptide mutant was grown according to the procedureused in Example 7. The bacterial cultures were also induced according tothe procedure used in Example 7. Luminescence was assayed and detectedaccording to the procedure used in Example 7.

FIG. 19 demonstrates that both cysteine and proline can be used as thefirst amino acid of NLpep and produce luminescence.

Example 13 Identification of the Optimal Composite Set of Mutations forthe Non-Luminescent Peptide

In the following examples, an optimal composite set(s) of mutations forthe non-luminescent peptide were identified. The mutant non-luminescentpeptides were generated as previously described in Example 1.

-   -   1) For non-luminescent peptide composite mutants NLpep53,        NLpep66, NLpep67, and NLpep68, a single colony of each was grown        according to the procedure used in Example 10. The bacterial        cultures were also induced according to the procedure used in        Example 10. TMR gel analysis and luminescence was assayed and        detected according to the procedure used in Example 10. The        results in FIG. 20 demonstrate the luminescence as well as        the E. coli expression of NLpeps containing multiple        mutations. 2) For non-luminescent peptide composite mutants        NLpep53 and NLpeps 66-74, a single colony of each was grown        according to the procedure used in Example 7. The bacterial        cultures were also induced according to the procedure used in        Example 7. Luminescence was assayed and detected according to        the procedure used in Example 7. The results in FIG. 21        demonstrate the luminescence of NLpeps containing multiple        mutations.    -   3) For non-luminescent peptide composite mutants NLpep53 and        NLpeps 66-76, a single colony of each was grown according to the        procedure used in Example 7. The bacterial cultures were also        induced according to the procedure used in Example 7.        Luminescence was assayed and detected according to the procedure        used in Example 7 except the non-luminescent polypeptide was 5A2        or 5A2+R11E (1:10 dilution of E. coli clarified lysate). The        results in FIG. 22 demonstrate the luminescence of NLpeps        containing multiple mutations with 5A2 or 5A2+R11E. These        results also demonstrate the lower luminescence when the NLpoly        mutation R11E is complemented with an NLpep containing E as the        9th residue (NLpep72, 75, and 76).    -   4) For non-luminescent peptide composite mutants NLpep1,        NLpep69, NLpep78 and NLpep79, a single colony of each was grown        according to the procedure used in Example 7. The bacterial        cultures were also induced according to the procedure used in        Example 7. Luminescence was assayed and detected according to        the procedure used in Example 7 except the non-luminescent        polypeptide was WT (1:10 dilution of E. coli clarified lysate).        The results in FIG. 23 demonstrate the luminescence of NLpeps        containing multiple mutations.

Example 14 Composite Non-Luminescent Polypeptide Mutants

In the following example, 9 mutations from the library screens werecombined into a composite clone (NLpoly1, SEQ ID NOS: 941,942), and thenone of the mutations reverted back to the original amino acid(NLpoly2-10, SEQ ID NOS: 943-960) in order to identify the optimalcomposite set. Based on previous results of NLpoly1-10, NLpoly11-13 (SEQID NOS: 961-966) were designed and tested for the same purpose. MutantNLpolys were generated as previously described in Example 1. Singlecolony of each non-luminescent polypeptide mutant was grown according tothe procedure used in Example 6. The bacterial cultures were alsoinduced according to the procedure used in Example 6. Luminescence wasassayed and detected according to the procedure used in Example 6 expectNLpep53 E. coli clarified lysate was used at 1:11.85 dilution.

FIG. 24 demonstrates the luminescence of NLpolys containing multiplemutations.

Example 15 Substrate Specificity of Non-Luminescent Polypeptide Mutants

The following example investigates the substrate specificity of thenon-luminescent polypeptide mutants. Luminescence generated fromluminescent complexes formed from various non-luminescent polypeptidemutants, either Furimazine or coelenterazine as a substrate, and variousnon-luminescent peptides.

HEK 293 cells were plated at 100,000 cells/ml into wells of a 24 wellplates containing 0.5 ml DMEM+10% FBS (50,000/well). The cells wereincubated in a 37° C., 5% CO₂ incubator overnight. DNA for expression ofeach non-luminescent polypeptide mutant was transfected in duplicate. 1ug plasmid DNA containing a non-luminescent polypeptide mutant was mixedwith OptiMEM (Life Technologies) to a final volume of 52 ul. 3.3 μl ofFugene HD (Promega Corporation) was added, and samples incubated for 15minutes at room temperature. 25 μl of each sample mixture was added totwo wells and incubated overnight in a 37° C., 5% CO₂ incubatorovernight. After overnight incubation, the growth media was removed and0.5 ml DMEM (without phenol red)+0.1% Prionex added. The cells were thenfrozen on dry ice (for how long) and thawed prior to detectingluminescence.

In FIGS. 25-26, luminescence was assayed and detected according to theprocedure used in Example 6, except NLpep53 E. coli clarified lysate wasused at 1:10 dilution and either Furimazine or coelenterazine in eitherNanoGlo Luciferase Assay buffer or DMEM were used. This datademonstrates the luminescence of NLpolys in NANOGLO and DMEM with eitherFurimazine or Coelenterazine as the substrate. This indicates thesubstrate specificity (Furimazine versus Coelenterazine) of the NLpolyin both NANOGLO and DMEM.

In FIG. 27, luminescence was assayed and detected according to theprocedure used in Example 6, except E. coli clarified lysate fromvarious non-luminescent peptides (NLpep1, NLpep9, NLpep48, NLpep53,NLpep69 or NLpep76) were used at 1:10 dilution. In addition, eitherFurimazine or coelenterazine in either NanoGlo Luciferase Assay bufferwere used. This data demonstrates the substrate specificity ofNLpoly/NLpep pairs.

In FIG. 28, luminescence was assayed and detected by separately dilutingNLpep53-HT fusion 1:10 and the non-luminescent polypeptide lysates 1:10in DMEM+0.1% Prionex. 20 μl of non-luminescent peptide and 20 μlnon-luminescent polypeptide were then combined and incubated for 10minutes at room temperature. 40 μl of NanoGlo Buffer with 100 uMFurimazine or DMEM with 0.1% Prionex and 20 uM Furimazine was then addedto the samples, and luminescence detected on GloMax Multi. This datademonstrates the substrate specificity of NLpolys expressed in HEK293cells.

In FIG. 29, luminescence was assayed and detected by separately dilutingNLpep1-HT, NLpep53-HT, NLpep69-HT or NLpep76-HT fusion 1:10 and thenon-luminescent polypeptide lysates 1:10 in DMEM+0.1% Prionex. 20 μl ofnon-luminescent peptide and 20 μl non-luminescent polypeptide were thencombined and incubated for 10 minutes at room temperature. 40 μl ofNanoGlo Buffer with 100 uM Furimazine or DMEM with 0.1% Prionex and 20uM Furimazine was then added to the samples, and luminescence detectedon GloMax Multi. This data demonstrates the luminescence of NLpolysexpressed in mammalian cells and assayed with various NLpeps.

Example 16 Signal-To-Background of Non-Luminescent Polypeptide Mutantswith Furimazine or Coelenterazine

The following example investigates signal-to-background of thenon-luminescent polypeptide mutants. Luminescence generated from variousnon-luminescent polypeptide mutants was measured using either Furimazineor coelenterazine as a substrate as well as with various non-luminescentpeptides.

HEK 293 cells were plated at 15,000 cells/well in 100 μl DMEM+10% FBSinto wells of 96-well plates. The cells were incubated in a 37° C., 5%CO₂ incubator overnight. Transfection complexes were prepared by adding0.66 ug each of plasmid DNA for expression of a non-luminescentpolypeptide mutant and a non-luminescent peptide mutant plasmid to afinal volume of 31 μl in OptiMem. 2 μl Fugene HD was added to eachtransfection complex and incubated for 15 minutes at room temperature.For each peptide/polypeptide combination, 5 μl of a transfection complexwas added to 6 wells of the 96-well plate and grown overnight at 37 C inCO₂ incubator. After overnight incubation, the growth media was removedand replaced with CO₂— independent media containing either 20 uMcoelenterazine or 20 uM Furimazine. The samples were incubated for 10minutes at 37° C., and kinetics measured over the course of 1 hour at37° C. on a GloMax Multi+. FIG. 30 demonstrates the substratespecificity of various NLpoly/NLpep pairs when the NLpoly is expressedin mammalian cells.

Example 17 Luminescence and Substrate Specificity

The following example investigates the luminescence and substratespecificity of various non-luminescent polypeptide mutants with NLpep69and using either Furimazine or coelenterazine as a substrate.

CHO cells were plated at 20,000 cells/well in 100 μl of DMEM+10% FBSinto wells of 96-well plates. The cells were incubated in a 37° C., 5%CO2 incubator overnight. Transfection complexes were prepared by adding0.66 ug each of plasmid DNA for expression of a non-luminescentpolypeptide mutant and a non-luminescent peptide mutant plasmid to afinal volume of 31 μl in OptiMem. 2 μl Fugene HD was added to eachtransfection complex and incubated for 15 minutes at room temperature.For each peptide/polypeptide combination, 5 μl of transfection complexwas added to 6 wells of the 96-well plate and grown overnight at 37 C inCO₂ incubator. After overnight incubation, the growth media was removedand replaced with CO₂— independent media containing either 20 uMcoelenterazine or 20 uM Furimazine. The samples were incubated for 10minutes at 37° C., and kinetics measured over the course of 1 hour at37° C. on a GloMax Multi+. FIG. 31 demonstrates the substratespecificity when NLpolys are coexpressed in mammalian cells withNLpep69.

Example 18 Luminescence and Substrate Specificity Between Live-Cell andLytic Conditions

The following example investigates the luminescence and substratespecificity of various non-luminescent polypeptide mutants with NLpep69,NLpep78 or NLpep79, using either Furimazine or coelenterazine as asubstrate and under either lytic or live cell conditions.

HEK 293 cells were plated at 15,000 cells/well in 100 μl DMEM+10% FBSinto wells of 96-well plates. The cells were incubated in a 37° C., 5%CO2 incubator overnight. Transfection complexes were prepared by adding0.66 ug each of plasmid DNA for expression of a non-luminescentpolypeptide mutant and a non-luminescent peptide mutant plasmid to afinal volume of 31 μl in OptiMem. 2 μl Fugene HD was added to eachtransfection complex and incubated for 15 minutes at room temperature.For each NLpoly-NLpep combination, 5 μl of transfection complex wasadded to 6 wells of the 96-well plate and grown overnight at 37 C in CO2incubator. After overnight incubation, the growth media was removed andreplaced with CO2-independent media containing either 20 uMcoelenterazine or 20 uM Furimazine. The samples were incubated for 10minutes at 37° C., and kinetics measured over the course of 1 hour at37° C. on a GloMax Multi+. FIGS. 32-34 demonstrate the substratespecificity of NLPolys coexpressed in mammalian cells with NLpep69, 78,or 79 in live-cell and lytic formats.

Example 19 Comparison of Non-Luminescent Polypeptide Mutants Expressedin E. coli

A single colony of each non-luminescent polypeptide was grown accordingto the procedure used in Example 7. The bacterial cultures were alsoinduced according to the procedure used in Example 7. Luminescence wasassayed and detected according to the procedure used in Example 7 exceptNLpep78-HT or NLpep79-HT at 1:1,000 dilution was used. FIG. 35demonstrates the luminescence of NLpolys expressed in E. coli andassayed with NLpep78 or 79.

Example 20 Ability of Non-Luminescent Polypeptide Clones to ProduceLuminescence without Complementing Non-Luminescent Peptide

A single colony of each non-luminescent polypeptide was grown accordingto the procedure used in Example 7. The bacterial cultures were alsoinduced according to the procedure used in Example 7. Luminescence wasassayed and detected according to the procedure used in Example 7 exceptno non-luminescent peptide was added to the assay buffer. FIG. 36demonstrates the luminescence of NLpolys expressed in E. coli andassayed in the absence of NLpep.

Example 21 Substrate Specificity of Non-Luminescent Polypeptide MutantsExpressed in E. coli

A single colony of each non-luminescent polypeptide was grown accordingto the procedure used in Example 7. The bacterial cultures were alsoinduced according to the procedure used in Example 7. Luminescence wasassayed and detected according to the procedure used in Example excepteither Furimazine or coelenterazine was mixed with NANOGLO Assay Buffer.FIG. 37 demonstrates the substrate specificity of NLpolys expressed inE. coli and assayed with NLpep78 or 79.

Example 22 Improved Luminescence of Non-Luminescent Polypeptide Mutantswith NLpep78

Complementation of the non-luminescent polypeptide mutants withNLpep78-HT was demonstrated in CHO and Hela cells.

CHO and Hela cells (CHO: 100,000 seeded the day prior to transfection;Hela: 50,000 seeded the day prior to transfection) were transfected with5 ng of a non-luminescent polypeptide mutant 5A2 or 5P or with wild-typenon-luminescent polypeptide using Fugene HD into wells of a 24-wellplate and incubated at 37° C. overnight. After the overnight incubation,the media was replaced with DMEM without phenol red, and the cellsfrozen at −80° C. for 30 minutes. The cells were then thawed andtransferred to a 1.5 ml tube. The cell lysates were then diluted 1:10DMEM without phenol red, 20 μl mixed with NLpep78 (NLpep78-HT7 E. colilysate diluted 1:1,000 in DMEM without phenol red) and shaken at roomtemperature for 10 minutes. 40 μl DMEM without phenol red and 20 uMFurimazine were added and luminescence measured on a GloMax with a 0.5second integration. FIG. 38 demonstrates the luminescence of NLpolysexpressed in mammalian cells and assayed with NLpep78.

Example 23 Non-Luminescent Polypeptide Fusions and NormalizingNon-Luminescent Polypeptide Concentrations

A comparison of raw and normalized luminescence from non-luminescentpolypeptide fused to either firefly luciferase (FIG. 39) or click beetlered luciferase (FIG. 40) were performed to provide insight into how muchbenefit, e.g., in expression, solubility and/or stability, stems fromthe concentration of the non-luminescent polypeptide as well ascomplementation as a fusion non-luminescent polypeptide.

HEK293, Hela or CHO cells were transfected with 5 ng 5P NLpoly-fireflyluciferase fusion, 5P NLpoly-click beetle luciferase fusion, wild-type5P-firefly luciferase fusion or wild-type 5P-click beetle luciferasefusion according to the procedure in Example 22. Lysates were alsoprepared according to Example 22. The cell lysates were then diluted1:10 DMEM without phenol red, 20 μl mixed with NLpep78 (diluted 1:100 inDMEM without phenol red; E. coli lysate) and shaken at room temperaturefor 10 minutes. 40 μl NanoGlo with 20 uM Furimazine or Bright-Glo(Promega Corporation) was added and luminescence measured on a GloMaxwith 0.5 second integration. FIGS. 39 and 40 demonstrate the specificactivity of 5P versus WT NLpoly expressed in mammalian cells and assayedwith NLpep78.

Example 24 Complementation in Live Cells

This example demonstrates complementation in live-cells using eitherwild-type or 5P NLpoly.Hela cells plated into wells of 96-well plated, transfected with 0.5 ngof wild-type or 5P non-luminescent polypeptide plasmid DNA using FugeneHD and incubated at 37° C. overnight. After the overnight incubation,the cells were then transfected with 0.5 ng NLpep78-HT plasmid DNA usingFugene HD and incubated at 37° C. for 3 hours. The media was thenreplaced with CO₂-independent media+0.1% FBS and 20 uM PBI-4377 andluminescence measured at 37° C. on a GloMax with 0.5 second integration.FIG. 41 demonstrates the live-cell complementation between 5P or WTNLpoly and NLpep78.

Example 25 Complementation in Cell-Free Extract

To demonstrate complementation in cell-free extract, 0.5 ug NLpep78-HTand 0.5 ug non-luminescent polypeptide mutant plasmid DNA were mixedwith TNT rabbit reticulocyte lysate master mix (Promega Corporation) andincubated at 30° C. for 1 hour. 25 μl of the cell-free expressionextract was mixed with 25 μl NanoGlo Luciferase Assay reagent andincubated at room temperature for 10 minutes. Luminescence was measuredon a GloMax with 0.5 second integration. FIG. 42 demonstratesluminescence from complementing NLpoly/NLpep pairs expressed in acell-free format.

Example 26 Binding Affinity of Non-Luminescent Polypeptide Expressed inMammalian Cells with Synthetic Non-Luminescent Peptide

To demonstrate the binding affinity between non-luminescent polypeptideand non-luminescent peptide pairs, non-luminescent polypeptide lysatesfrom Hela, HEK293 and CHO cells were prepared as previously describedand diluted 1:10 PBS+0.1% Prionex. 4× concentrations of non-luminescentpeptide (synthetic) were made in PBS+0.1% Prionex. 20 μl of thenon-luminescent polypeptide lysate was mixed with 20 μl non-luminescentpeptide and shaken at room temperature for 10 minutes. 40 μl of NanoGloLuciferase Assay Reagent or PBS+0.1% Prionex with Furimazine was addedand shaken at room temperature for 10 minutes. Luminescence was detectedon a GloMax with 0.5 s integration. Kd values were determined usingGraphpad Prism, One Site-Specific Binding. FIGS. 43 and 44 demonstratethe dissociation constants measured under various buffer conditions (PBSfor complementation then NanoGlo for detection, PBS for complementationand detection, NanoGlo for complementation and detection).

Example 27 Improved Binding Affinity when Cysteine Mutated toPhenylalanine in Non-Luminescent Peptide Mutants

To demonstrate improved binding affinity in non-luminescent peptidemutants with a mutated cysteine at the 8th residue of the peptide,non-luminescent polypeptide mutant lysates from Hela, HEK293 and CHOcells were prepared as previously described and diluted 1:10 PBS+0.1%Prionex. 4× concentrations of non-luminescent peptide (NLpep) were madein PBS+0.1% Prionex+10 mM DTT. 20 μl of the non-luminescent polypeptidelysate was mixed with 20 μl non-luminescent peptide and shaken at roomtemperature for 10 minutes. 40 μl of NanoGlo Luciferase Assay Reagentwas added and shaken at room temperature for 10 minutes. Luminescencewas detected on a GloMax with 0.5 s integration. FIG. 45 demonstratesNLpep C8F mutation significantly improves the binding affinity for 5P.

Example 28 Detectable Luminescence of Polypeptide Variants withoutNon-Luminescent Peptide in Hela Cells

To demonstrate luminescence in non-luminescent polypeptide withoutnon-luminescent peptide, Hela cells (10,000 seeded the day prior totransfection) in wells of a 96-well plate were transfected with varyingamounts of non-luminescent polypeptide+pGEM-3zf Carrier DNA to a totalof 50 ng using Fugene HD and incubated 37° C. overnight. Afterincubation, the media was replaced with CO₂-independent media+0.1%FBS+20 uM Furimazine and incubated at 37° C. for 10 minutes, andluminescence detected on a GloMax with 0.5 s integration. FIG. 46demonstrates the luminescence of NLpoly WT or 5P in live Hela cellswithout NLpep after transfection of various amounts of plasmid DNA.

Example 29 Generation of Additional Non-Luminescent Polypeptide Variants

Additional non-luminescent polypeptide variants: Ile-11 (Ile at residue11), Val-11, Tyr-11, Glu-11, Glu-157, Pro-157, Asp-157, Ser-157,Met-149, Leu-106, NLpoly11, and NLpoly12 were generated as describedbelow, and their expression analyzed. The additional non-luminescentpolypeptide variants were made in the 5A2 non-luminescent polypeptidebackground.

Fresh individual colonies (KRX) of each additional non-luminescentpolypeptide variants were picked and grown overnight in LB+ampicillin(100 ug/ml) at 30° C. and then diluted 1:100 in LB+ampicillin and grownat 37° C. for 2.5 hours (OD600 ˜0.5). Rhamnose was added to a finalconcentration of 0.2%, and the cells were split in triplicate and grownovernight at 25° C. for ˜18 h. Cells were lysed using 0.5× Fast Breakfor 30 minutes at ambient temperature, snap-frozen on dry ice, andstored at −20° C. Upon fast thawing, soluble fractions were prepared bycentrifugation at 10K for 15 min at 4° C. Samples were assayed forluminescence on a Tecan Infinite F-500 luminometer.

FIG. 49 demonstrates that total lysate and soluble fraction of eachnon-luminescent polypeptide variant as analyzed by SDS-PAGE. The dataprovides information about expression, solubility and stability of theadditional non-luminescent polypeptide variants. A majority of theadditional non-luminescent polypeptide variants produced more protein(total and soluble) than wild-type, but in many cases, the difference issubtle. Improved expression for NLpoly11 and NLpoly12 was morenoticeable.

Example 30 Background Luminescence of Additional Non-LuminescentPolypeptide Variants

The background luminescence of the additional non-luminescentpolypeptide variants generated in Example 29 was measured by incubating25 μl of non-luminescent polypeptide variant lysate with 25 μl DMEM atroom temperature for 10 minutes. 50 μl NanoGlo Luciferase Assay Reagentwas then added, and luminescence measured at 5 and 30 minutes on a TecanInfinite F500. NLpep53 (Pep 53) alone and DMEM (DMEM) alone were used ascontrols. FIG. 47 demonstrates that a majority of the additionalnon-luminescent polypeptide variants showed elevated backgroundluminescence.

Example 31 Luminescence of Additional Non-Luminescent PolypeptideVariants after Complementation

Luminescence of the additional non-luminescent polypeptide variantsgenerated in Example 28 was measured by incubating 25 μl ofnon-luminescent polypeptide variant lysate with 25 μl NLpep-53 at roomtemperature for 10 minutes 50 μl NanoGlo Luciferase Assay Reagent wasthen added, and luminescence measured at 5 and 30 minutes on a TecanInfinite F500. NLpep53 (Pep 53) alone and DMEM (DMEM) alone were used ascontrols. FIG. 48 demonstrates that the non-luminescent polypeptidevariants Val-11, Glu-11, Glu-157, Pro-157, Asp-157, Ser-157 and Met-149generated significantly more luminescence than parental 5A2.

Example 32 Correlation Between Increased Background Luminescence ofNon-Luminescent Polypeptide in the Absence of Non-Luminescent Peptideand Amount of Protein in Soluble Fraction

Individual colonies of the non-luminescent polypeptide variants 3P, 3E,5P, 5E, 6P and 6E were picked and grown overnight in LB+ampicillin at30° C. and then diluted 1:100 in LB+ampicillin and grown at 37° C. for2.5 hours (OD600 ˜0.5). Rhamnose was added to a final concentration of0.2%, and the cells were split in triplicate and grown overnight at 25°C. for ˜18 h. Cells were lysed using 0.5× Fast Break for 30 minutes atambient temperature, snap-frozen on dry ice, and stored at −20° C. Uponfast thawing, soluble fractions were prepared by centrifugation at 10Kfor 15 min at 4° C. Samples were assayed for luminescence on a TecanInfinite F-500. FIG. 50A shows the total lysate and soluble fraction ofeach non-luminescent polypeptide variant. FIG. 50B shows the backgroundluminescence of each non-luminescent polypeptide variant. FIG. 51 showsthe luminescence generated with each non-luminescent polypeptide variantwhen complemented with 10 or 100 nM NLpep78 (NVSGWRLFKKISN) in LBmedium.

Example 33 Elongations and Deletions of Non-Luminescent Polypeptide

The non-luminescent polypeptide variant 5P was either elongated at theC-terminus by the addition of the residues VAT, AA, VTG, VT, VTGWR (SEQID NO: 2576), VTGW (SEQ ID NO: 2577), V, A, VA, GG, AT, GTA, ATG or GTor deletion of 1 to 7 residues at the C-terminus of 5P, e.g.,D1=deletion of 1 residue, D2=deletion of 2 residues, etc. Backgroundluminescence in E. coli lysates (FIG. 52) and luminescence generatedafter complementation with NLpep78 (FIG. 53; NVSGWRLFKKISN (SEQ ID NO:374)) or NLpep79 (FIG. 54; NVTGYRLFKKISN(SEQ ID NO: 376)) were measured.FIG. 55 shows the signal-to-background of the non-luminescentpolypeptide 5P variants. FIG. 56 provides a summary of the luminescentresults. FIG. 57 shows the amount of total lysate and soluble fractionin each non-luminescent polypeptide 5P variant.

Example 34 Comparison of 5P and I107L Non-Luminescent PolypeptideVariant

FIG. 58 shows the amount of total lysate and soluble fraction of 5P andI107L (A), luminescence generated by 5P or I107L without non-luminescentpeptide or with NLpep78 or NLpep79 (B) and the improvedsignal-to-background of I107L over 5P (C).

Example 35 Generation of 5P Non-Luminescent Polypeptide Mutants

Mutations identified in a screening of random mutations in the 5Pnon-luminescent polypeptide variant were generated as previouslydescribed. Each single 5P non-luminescent polypeptide mutant colony wasinoculated in 200 μl Minimal Media and incubated with shaking at 37° C.for 20 hours. 10 μl of the culture was then added to 190 μl of freshMinimal Media and incubated again with shaking at 37° C. for 20 hours.10 μl of the second culture was then added to 190 μl Auto-InductionMedia (Minimal Media+5% glucose+2% rhamnose) and incubated with shakingat 25° C. for 18 hours to allow expression of the non-luminescentpolypeptide mutant. 10 μl of the 5P non-luminescent polypeptide mutantexpression culture was added to 40 μl of assay lysis buffer containingNLpep78-HT (1:386 dilution) or NLpep79-HT (1:1,000 dilution) and shakenat room temperature for 10 minutes. 50 μl of NanoGlo Assay Buffercontaining 100 uM coelenterazine was added and shaken at roomtemperature for 10 minutes. Luminescence was measured on GloMax with 0.5sec integration. FIGS. 59-62A shows background luminescence while FIGS.59-62B and C show luminescence generated after complementation withNLpep78 or NLpep79.

Example 36 Binding Affinity Between Elongated Non-LuminescentPolypeptide Variant and Deleted Non-Luminescent Peptide

The binding affinity between an elongated non-luminescent polypeptidevariant, i.e., containing additional amino acids at the C-terminus, anda deleted non-luminescent peptide, i.e., deleted amino acids at theN-terminus.

Lysates of E. coli expressing non-luminescent polypeptide 5P/+V/+VT/+VTGprepared as previously described were diluted 1:2000 in PBS+0.1%Prionex. 25 μl of the diluted lysate was incubated with 25 μl ofNLpep78, NLpep80, NLpep81 or NLpep82 (diluted 0-500 nM in dilutionbuffer) for 5 min at room temp. 50 μl of Furimazine diluted to 1× withNanoGlo Assay Buffer was added to each sample and incubated for 10minutes at room temperature. Luminescence was measured on a GloMax Multiwith 0.5 s integration time. FIG. 63 demonstrates the binding affinitybetween NLpolys with additional amino acids at the C-terminus withNLpeps with amino acids deleted from the N-terminus.

Example 37 Binding Affinity Between Non-Luminescent PolypeptideExpressed in E. coli and Synthetic Non-Luminescent Peptide

Non-luminescent polypeptide LB lysates were prepared and diluted 1:100into PBS+0.1% Prionex. 2× dilutions of synthetic NLpep78 were made inPBS+0.1% Prionex. 25 μl of the diluted non-luminescent polypeptidelysate was mixed with 25 μl of each dilution of non-luminescent peptideand incubated 3 minutes at ambient temperature. 50 μl of NanoGloLuciferase Assay Reagent was added, incubated for 5 minutes at roomtemperature, and luminescence measured on a GloMax Multi+. FIG. 64 showsthe calculated Kd values using one-site specific binding.

Example 38 Binding Affinity Between SP Non-Luminescent PolypeptideExpressed in Mammalian Cells and NLpep80 or NLpep87

Lysates of CHO, HEK293T, or HeLa cells expressing NLpoly 5P were diluted1:1000 in dilution buffer (PBS+0.1% Prionex.) 25 μl of diluted lysatewas incubated with 25 μl of NLpep80/87 (diluted 0-5 μM in dilutionbuffer) for 5 min at room temp. 50 μl of furimazine (diluted to 1× withNanoGlo buffer) was added to each well, and the plate was incubated for10 min at room temp. Luminescence was then read on a GloMax Multi with0.5 s integration time (FIG. 65).

Example 39 Binding Affinity Between 5P Non-Luminescent PolypeptideExpressed in E. coli and NLpep80 or NLpep87

Lysates of E. coli expressing NLpoly 5P were diluted 1:2000 in dilutionbuffer (PBS+0.1% Prionex.) 25 μl of diluted lysate was incubated with 25μl of NLpep80/87 (diluted 0-5 μM in dilution buffer) for 5 min at roomtemp. 50 μl of furimazine (diluted to 1× with NanoGlo buffer) was addedto each well, and the plate was incubated for 10 min at room temp.Luminescence was then read on a GloMax Multi with 0.5 s integration time(FIG. 66).

Example 40 Complementation Between a Deleted Non-Luminescent Polypeptideand Elongated Non-Luminescent Peptide

Complementation between a deleted non-luminescent polypeptide, i.e.,amino acids deleted from the C-terminus, and an elongatednon-luminescent peptide, i.e., amino acids added to the N-terminus, wasperformed. NLpep-HT E. coli clarified lysates as prepared as previouslydescribed in Example 6. The amount of NLpep-HT was quantitated via theHaloTag fusion. Briefly, 10 μl of clarified lysate was mixed with 10 μlHaloTag-TMR ligand (diluted 1:100) and 80 μl water and incubated at roomtemperature for 10 minutes. 33.3 μl 4×SDS Loading Buffer was added andincubated at 95° C. for 5 minutes. 15 μl was loaded onto an SDS-PAGE geland imaged on a Typhoon. Based on the intensities from the SDS-PAGE gel,non-luminescent peptides were diluted in PBS+0.1% Prionexnon-luminescent peptides to make equivalent concentrations. Thenon-luminescent polypeptide lysates were then diluted 1:100 in PBS+0.1%Prionex. 20 μl of diluted non-luminescent polypeptide and 20 μl dilutednon-luminescent peptide were mixed and shaken at room temperature for 10minutes. 40 μl NanoGlo Luciferase Assay Reagent was added and shaken atroom temperature for 10 minutes. Luminescence was measured on a GloMaxusing 0.5 sec integration. FIG. 67 demonstrates the luminescence ofNLpolys with amino acids removed from the C-terminus with NLpeps withadditional amino acids on the N-terminus.

Example 41 Binding Affinity Between 5P Non-Luminescent PolypeptideExpressed in Hela Cells and NLpep78 or Truncated NLpep78 (NLpep80-87)

5P non-luminescent polypeptide lysate was prepared from Hela cells aspreviously described and diluted prepared 1:10 in PBS+0.1% Prionex. 4×concentrations (range determined in preliminary titration experiment) ofnon-luminescent peptide (synthetic peptide; by Peptide 2.0 (Virginia);made at either 5, 10, or 20 mg scale; blocked at the ends by acetylationand amidation, and verified by net peptide content analysis) wasprepared in PBS+0.1% Prionex. 20 μl 5P non-luminescent polypeptide and20 μl non-luminescent peptide were mixed and shaken at room temperaturefor 10 minutes. 40 μl of NanoGlo Luciferase Assay reagent was added andshaken at room temperature for 10 minutes. Luminescence was measured onGloMax with 0.5 s integration. FIG. 68 demonstrates the binding affinityand corresponding luminescence between 5P and truncated versions ofNLpep78. The binding affinity is increased when 1 amino acid is removedfrom the N-terminus, the C-terminus, or 1 amino acid from each terminus.Removing more than 1 amino acid from either terminus lowers the affinitybut does not always lower the Vmax to the same extent.

Example 42 Binding Affinity Between Elongated Non-LuminescentPolypeptide and Truncated Non-Luminescent Peptide

The binding affinity between an elongated non-luminescent polypeptide,i.e., one with 2 extra amino acids on C-terminus, and a truncatednon-luminescent peptide, i.e., one with 2 amino acids removed fromN-terminus (NLpep81), was determined.

Non-luminescent polypeptide lysate was prepared as previously describedand diluted prepared 1:100 in PBS+0.1% Prionex. 2× dilutions of NLpep81(synthetic peptide; by Peptide 2.0 (Virginia); made at either 5, 10, or20 mg scale; blocked at the ends by acetylation and amidation, andverified by net peptide content analysis) was prepared in PBS+0.1%Prionex. 25 μl non-luminescent polypeptide and 25 μl of eachnon-luminescent peptide dilution were mixed and shaken at roomtemperature for 3 minutes. 50 μl of NanoGlo Luciferase Assay reagent wasadded and shaken at room temperature for 5 minutes.

Luminescence was measured on GloMax with 0.5 s integration. FIG. 69shows the calculate Kd values using one-site specific binding.

Example 43 Binding Affinity Between Elongated Non-LuminescentPolypeptide and Truncated Non-Luminescent Peptide

The binding affinity between an elongated non-luminescent polypeptide,i.e., one with 3 extra amino acids on C-terminus, and a truncatednon-luminescent peptide, i.e., one with 3 amino acids removed fromN-terminus (NLpep82), was determined.

Non-luminescent polypeptide lysate was prepared and diluted prepared1:100 in PBS+0.1% Prionex. 2× dilutions of NLpep82 (synthetic peptide;by Peptide 2.0 (Virginia); made at either 5, 10, or 20 mg scale; blockedat the ends by acetylation and amidation, and verified by net peptidecontent analysis) was prepared in PBS+0.1% Prionex. 25 μlnon-luminescent polypeptide and 25 μl of each non-luminescent peptidedilution were mixed and shaken at room temperature for 3 minutes. 50 μlof NanoGlo Luciferase Assay reagent was added and shaken at roomtemperature for 5 minutes. Luminescence was measured on GloMax with 0.5s integration. FIG. 70 shows the calculate Kd values derived usingone-site specific binding.

Example 44 Binding Affinity Between Non-Luminescent Polypeptide ClonesExpressed in E. coli and Synthetic NLpep78

Non-luminescent polypeptide variants were grown in M9 minimal media.Individual colonies were inoculated and grown overnight at 37° C.Samples were diluted 1:20 in M9 minimal media and grown overnight at 37°C. Samples were again diluted 1:20 in M9 induction media and grownovernight at 25° C. Samples were pooled, and 100 μl of the pooled cellswere lysed with 400 μl of PLB lysis buffer and incubate at roomtemperature for 10 minutes. The lysates were diluted 1:100 in PBS+0.1%Prionex. 2× dilutions of synthetic NLpep78 were made in PBS+0.1%Prionex. 25 μl of non-luminescent polypeptide dilution was mixed with 25μl of each non-luminescent peptide dilution and incubated for 3 minutesat room temperature. 50 μl of NanoGlo Luciferase Assay Reagent wasadded, incubated at room temperature for 5 minutes, and luminescenceread on GloMax Multi+. FIG. 71 shows the calculate Kd values derivedusing one-site specific binding.

Example 45 Determination of the Effect of Mutations on Km

Using diluted pooled lysates from Example 11, 25 μl of non-luminescentpolypeptide diluted lysate (1:100 in PBS+0.1% Prionex) was mixed with 25μl of 500 nM NLpep78 for each sample and incubated at room temperaturefor 5 minutes. 2× dilutions of Furimazine in NanoGlo Luciferase AssayBuffer were prepared, and 50 μl of non-luminescent peptide andnon-luminescent polypeptide sample mixed with 50 μl ofNanoGlo/Furimazine dilutions. Luminescence was measured after 5 minuteincubation at room temperature. FIG. 72 show the calculated Km derivedusing Michaelis-Menten.

Example 46 Demonstration of a Three-Component Complementation

A tertiary complementation using 2 NLpeps and NLpoly 5P non-luminescentpolypeptide is demonstrated. NLpoly 5P-B9 (5P with residues 147-157deleted) and NLpep B9-HT (Met+residues 147-157 fused to N-terminus ofHT7) lysates were prepared.

A) NLpoly 5P-B9+NLpoly B9 Titration with NLpep78

NLpoly 5P-B9+NLpoly B9 was titrated with NLpep78. 20 μl 5P-B9(undiluted) was mixed with 20 μl peptideB9-HT (undiluted). Dilutions ofNLpep78 (synthetic peptide, highest concentration=100 uM) were made inPBS+0.1% Prionex. 20 μl NLpep78 was added to 40 μl of the5P-B9+peptideB9-HT mixture and shaken at room temperature for 10minutes. 60 μl NanoGlo Luciferase Assay Reagent was added and shaken atroom temperature for 10 minutes. Luminescence was measured on GloMaxwith 0.5 s integration.

B) NLpoly 5P-B9+NLpep78 Titration with NLpepB9-HT

20 μl NLpoly 5P-B9 (undiluted) was mixed with 20 μl NLpep78 (100 uM).Dilutions of peptideB9-HT (highest concentration=undiluted) were made inPBS+0.1% Prionex. 20 μl of peptideB9-HT was added to 40 μl of the5P-B9+NLpep78 mixture and shaken at room temperature for 10 minutes. 60μl NanoGlo Luciferase Assay Reagent was added and shaken at roomtemperature for 10 minutes. Luminescence was measured on GloMax with 0.5s integration.

FIG. 73 demonstrates the feasibility of a ternary system consisting of 2different NLpeps and a truncated NLpoly. Since all 3 components arenon-luminescent without the other 2, this system could be configuredsuch that each NLpep is fused (synthetically or genetic engineering) toa binding moiety and the truncated NLpoly used at high concentrations toproduce light only in the presence of an interaction between the bindingmoieties, or such that each of the 3 components are fused to bindingmoieties to produce light only in the event of ternary complexformation.

Example 47 Complementation with NLpep88 (NLpep78 with Gly as 6th ResidueInstead of Arg)

NLpep88-HT and 5P E. coli clarified lysates were prepared as previouslydescribed. Serial dilutions of NLpep88-HT lysate were made in PBS+0.1%Prionex. 20 μl of 5P lysate and 20 μl NLpep88-HT lysate were mixed andshaken at room temperature for 10 minutes. 40 μl of NanoGlo LuciferaseAssay Reagent was added and shaken at room temperature for 10 minutes.Luminescence was measured on GloMax with 0.5 s integration. FIG. 74demonstrates the importance of the arginine residue at the 6th positionof the NLpep. While there is no increase in luminescence above 5P aloneat lower concentrations of NLpep88, high concentrations of NLpepincreased the luminescence suggesting a catalytically compromisedcomplex and not a lack of interaction between 5P and NLpep88.

Example 48 Subcellular Localization of NLpep78 and 79 as N-TerminalFusions to HaloTag

U2OS cells were plated and left to recover overnight at 37° C. Cellswere then transfected with HaloTag alone DNA construct or theHaloTag-NanoLuc peptide DNA constructs (all under the control of CMVpromoter): P1-HT, P78-HT or P79-HT diluted 1:10 with carrier DNA (pSI)using FuGENE HD and incubated for 24 hours at 37° C. Cells were thenlabeled with HaloTag-TMR ligand by the manufacturer's standard rapidlabeling protocol and imaged. FIG. 75 demonstrates that NLpep78 and 79do not alter the intracellular localization of the HaloTag protein.

Example 49 Subcellular Localization of Non-Luminescent Polypeptide (WTand 5P)

U2OS cells were plated and left to recover overnight at 37° C. Cellswere either kept as non-transfection controls or transfected with theNanoLuc DNA constructs: FL, NLpoly (wt) or NLpoly(5P) diluted 1:10 withcarrier DNA (pSI) using FuGENE HD and incubated for 24 hours at roomtemperature. Cells were fixed and subsequently processed for ICC. ICCwas done using 1:5000 GS (PRO) primary antibody overnight at 4° C.followed by an Alexa488 goat anti-rabbit secondary antibody. FIG. 76demonstrates that both NLpoly WT and NLpoly 5P localize uniformly incells.

Example 50 Demonstration that Non-Luminescent Polypeptide can Easily andQuickly Detect Non-Luminescent Peptide Conjugated to a Protein ofInterest

99 μl of NLpep53-HT E. coli clarified lysate was mixed with 24.75 μl4×SDS loading buffer. 1:10 serial dilutions of the lysate-loading buffermixture were made and incubated at 95° C. for 5 minutes. 15 μl wasloaded onto a SDS-PAGE gel. After gel completions, it was transferred toPVDF using iBlot and washed with 10 mL NLpoly L149M E. coli clarifiedlysate at room temperature for 30 minutes. The membrane was then placedon a LAS4000 imager and 2 mL NanoGlo® Luciferase Assay Reagent added. A60 second exposure was taken (FIG. 77).

Example 51 Site Saturation at Non-Luminescent Polypeptide Positions 31,46, 108, 144, and 157 in the Context of 5P

Single amino acid change variants were constructed onto NLpoly 5P (pF4Agvector background) at the sites according to table 5 below. In effect,the native residue was varied to each of the 19 alternative amino acidsfor a total of 95 variants.

TABLE 5 Position 31 Position 46 Position 108 Position 144 Position 157B1 Ala E3 Ala H5 Ala C8 Ala F10 Ala C1 Cys F3 Cys A6 Cys D8 Cys G10 CysD1 Asp G3 Asp B6 Asp E8 Asp H10 Asp E1 Glu H3 Glu C6 Glu F8 Glu A11 GluF1 Gly A4 Phe D6 Phe G8 Phe B11 Phe G1 His B4 Gly E6 Gly H8 Gly C11 GlyH1 Ile C4 His F6 His A9 His D11 His A2 Lys D4 Ile G6 Ile B9 Ile E11 IleB2 Leu E4 Lys H6 Lys C9 Lys F11 Lys C2 Met F4 Met A7 Leu D9 Leu G11 LeuD2 Asn G4 Asn B7 Met E9 Met H11 Met E2 Pro H4 Pro C7 Pro F9 Asn A12 AsnF2 Gln A5 Gln D7 Gln G9 Pro B12 Gln G2 Arg B5 Arg E7 Arg H9 Gln C12 ArgH2 Ser C5 Ser F7 Ser A10 Arg D12 Ser A3 Thr D5 Thr G7 Thr B10 Ser E12Thr B3 Val E5 Val H7 Val C10 Val F12 Val C3 Trp F5 Trp A8 Trp D10 TrpG12 Trp D3 Tyr G5 Tyr B8 Tyr E10 Tyr H12 Tyr

Individual colonies were grown in LB+amp and incubated overnight at 30°C. A 5P control was also included. The overnight cultures were used toinoculate fresh LB+amp (1:100), and these cultures grew for 2 hours 45minutes at 37° C. Rhamnose was added to 0.2%, and the cultures left togrow/induce overnight at 25° C. After 18 hours of induction, cells werelysed using 0.5× FastBreak (30 min ambient temperature), snap frozen ondry ice, and stored at −20° C. Following a fast thaw, samples wereassayed in the absence and presence of Pep87 (aka NLpep 87).

For the (−) peptide reactions, 30 uL lysate was incubated with 30 uL PBSpH 7.5 for 10 min and then 60 uL NanoGlo® Luciferase Assay reagent(Promega Corporation) added. After 5 minutes, luminescence was measured.For the (+) peptide reactions, 30 uL lysate was incubated with 30 uL of8 nM Pep87. After 10 min, 60 uL NanoGlo® Luciferase Assay reagent wasadded, and luminescence measured at 5 minutes.

Luminescence (RLU) data for the (−) peptide samples were normalized tothe readings for the 5P control, and these results are presented in FIG.78. Luminescence (RLU) data for the (+) peptide samples were alsonormalized to 5P, but then also normalized to the values in FIG. 76 inorder to represent signal to background (S/B; FIG. 79).

Example 52 Use of the High Affinity Between NLpoly and NLpep for ProteinPurification/Pull Downs

MAGNEHALOTAG beads (Promega Corporation; G728A) were equilibrated asfollows:

a) 1 mL of beads were placed on magnet for ˜30 sec, and the bufferremoved; b) the beads were removed from magnet, resuspended in 1 mLPBS+0.1% Prionex, and shaken for 5 min at RT; and c) steps a) and b)were repeated two more times NLpep78-HaloTag (E. coli clarified lysate)was bound to MAGNEHALOTAG beads by resuspending the beads in 1 mLNLpep78-HT clarified lysate, shaking for 1 hr at RT and placing onmagnet for ˜30 sec. The lysate (flow through) was removed and saved foranalysis. NLpoly 8S (E. coli clarified lysate) was bound to the NLpep78bound-MagneHaloTag beads from the step above by resuspending the beadsin 1.5 mL 8S lysate, shaking for 1 hr at RT and placing on a magnet for˜30 sec. The lysate (flow through) was removed and saved for analysis.The beads were resuspended in 1 mL PBS+0.1% Prionex, shaken for 5 min atRT, placed on magnet for ˜30 sec, and PBS (wash) removed. The beads werewashed three more times.

To elute the bound peptide/polypeptide, the beads were resuspended in500 uL 1×SDS buffer and shaken for 5 min at RT. The beads were thenplaced on a magnet for ˜30 sec; the SDS buffer (elution) removed andsaved for analysis. The elution was repeated one more time.

The samples were then analyzed by gel. 37.5 uL of sample (exceptelutions) was mixed with 12.5 uL 4×SDS buffer and incubated at 95° C.for 5 min. 5 uL was loaded onto a Novex 4-20% Tris-Glycine gel and runat −180V for ˜50 min. The gel was stained with SimplyBlue Safe Stain andimaged on a LAS4000 imager.

FIG. 94 illustrates that the affinity of NLpoly and NLpep is sufficientto allow for purification from an E. coli lysate. As NLpoly 8S waspurified from an E. coli lysate, it is reasonable to expect a proteinfused to NLpoly 8S (or other variant described herein) could also bepurified in a similar fashion. While in this example the NLpep wasimmobilized and used to purify NLpoly, it is also reasonable to expect asimilar result if NLpoly were immobilized.

Example 53 Kinetics of NLpoly/NLpep Binding

2× concentrations of synthetic NLpep were made and diluted 2.7-fold ninetimes (10 concentrations) in PBS+0.1% Prionex. Final concentrations usedin the assay were 30 uM-3.9 nM. WT NLpoly (E. coli clarified lysate;1:10,000) or 11S (1:10,000,000) was diluted in NanoGlo+100 uM Furmazine(Fz). 50 uL of NLpep was placed into wells of white 96-well assay plate.50 uL NLpoly/NanoGlo/Fz was injected into the wells using the injectoron GloMax® Multi+ instrument, and luminescence measured every 3 sec over5 min. k_(obs) was found by fitting data to: Y=Y_(max)(1−e^(−k) ^(obs)^(t)) using Graphpad Prism. k_(on) and k_(off) were then fitted to:k_(obs)=[NLpep]k_(on)+k_(off). FIG. 95 illustrates the association anddissociation rate constants for the binding between NLpolys and NLpeps.

Example 54 NLpoly/NLpep Substrate Affinity

NLpoly was diluted into PBS+0.1% Prionex as follows: WT at 1:10⁵, 5P at1:10′, and 11S at 1:10⁸. NLpep was diluted into PBS+0.1% Prionex asfollows: 30 uM for WT NLpoly studies or 3 uM for NLpoly 5P and 11Sstudies. 50 uL NLpoly/NLpep was incubated at RT for 5 min, 50 uLNanoGlo+Fz (ranging from 100 uM to 1.2 uM, 2×) added, and incubated for10 min at RT. Luminescence was measured on GloMax® Multi+ with 0.5 secintegration. Km was derived using Graphpad Prism, Michaelis-Mentonbest-fit values. FIG. 96 illustrates the Km values for variousNLpoly/NLpep pairs.

Example 55 Substrate Effect on NLpoly/NLpep Affinity

11S (E. coli clarified lysate) was diluted into PBS+0.1% Prionex at1:10⁷. Synthetic NLpep79 was diluted serially (1:2) from 800 nM to 0.39nM (2×). 20 uL 11S+20 uL NLpep79 were then mixed and incubated for 5 minat RT. 40 uL NanoGlo+5 uM or 50 uM Fz was added and incubated another 5min at RT. Luminescence was measured on GloMax® Multi+ with 0.5 secintegration. Kd was derived using Graphpad prism, One site-Specificbinding value. FIG. 97 illustrates that saturating concentrations offurimazine increase the affinity between 11S and NLpep79.

Example 56 Km for NLpoly 5A2:NLpep

NLpoly 5A2 was diluted into PBS+0.1% Prionex at 1:10⁵. NLpep (WT, NLpep78 or NLpep79) was diluted into PBS+0.1% Prionex to 30 uM. 50 uLNLpoly/NLpep was incubated at RT for 5 min. 50 uL NanoGlo+Fz (rangingfrom 100 uM to 1.2 uM, 2×) was added and incubated for 10 min at RT.Luminescence was measured on GloMax® Multi+ with 0.5 sec integration. Kmwas derived using Graphpad Prism, Michaelis-Menton best-fit values. FIG.98 illustrates the Km values for NLpoly5A2 and NLpep WT, 78, and 79.

Example 57 Luminescence of NLpoly without NLpep

E. coli clarified lysate were prepared as described previously forNLpoly WT, 5A2, 5P, 8S and 11S. 50 uL of each lysate and 50 uLNanoGlo+Fz were mixed and incubated for 5 min RT. Luminescence wasmeasured on GloMax® Multi+ with 0.5 sec integration. FIG. 99 illustratesthat the ability of the NLpoly to produce luminescence in the absence ofNLpep gradually increased throughout the evolution process resulting in˜500 fold higher luminescence for 11S than WT NLpoly.

Example 58 Improved Luminescence in E. coli Throughout Evolution Process

A single NLpoly colony of WT, 5A2, 5P, 8S or 11S was inoculated in 200uL minimal media and grown for 20 hrs at 37° C. on shaker. 10 uL of theovernight culture was diluted into 190 uL fresh minimal media and grownfor 20 hrs at 37° C. on shaker. 10 uL of this overnight culture wasdiluted into 190 uL auto-induction media (previously described) andgrown for 18 hrs at 25° C. on shaker. The auto-induced cultures werediluted 50-fold (4 uL into 196 uL assay lysis buffer), 10 uL expressionculture added to 40 uL of assay lysis buffer containing NLpep(synthetic; 1 nM; WT, NLpep78, NL79 or NLpep80) and shaken for 10 min atRT. 50 uL NanoGlo+Fz was added, and samples shaken for 5 min at RT.Luminescence was measured on a GloMax luminometer with 0.5 secintegration. FIG. 100 illustrates the improvement in luminescence fromE. coli-derived NLpoly over the course of the evolution process, anoverall ˜10⁵ improvement (from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).

Example 59 Improved Luminescence in HeLa Cells Throughout EvolutionProcess

50 ng plasmid DNA expressing NLpoly WT, 5A2, 5P, 8S or 11S wastransfected into HeLa cells into wells of a 12-well plate usingFugeneHD. The cells were then incubated overnight at 37° C./5% CO₂. Themedia was replaced with 500 uL DMEM without phenol red, and the cellsfrozen at −80° C. for >30 min. The cells were thawed and transferred to1.5 mL tubes.

NLpep WT, NLpep78, NLpep79 or NLpep 80 (synthetic) were diluted to 10 nMin PBS+0.1% Prionex, and 25 μl mixed with 25 uL of each of the NLpolycell lysate. The samples were shaken for 10 min at RT, and then 50 uLNanoGlo+100 uM Fz added and incubated for 5 min at RT. Luminescence wasmeasured on a GloMax luminometer with 0.5 s integration. FIG. 101illustrates the improvement in luminescence from HeLa-expressed NLpolyover the course of the evolution process, an overall ˜10⁵ improvement(from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).

Example 60 Improved Luminescence in HEK293 Cells Throughout EvolutionProcess

50 ng plasmid DNA expressing NLpoly WT, 5A2, 5P, 8S or 11S wastransfected into HEK293 cells into wells of a 12-well plate usingFugeneHD. The cells were then incubated overnight at 37° C./5% CO2. Themedia was replaced with 500 uL DMEM without phenol red, and the cellsfrozen at −80° C. for >30 min. The cells were thawed and transferred to1.5 mL tubes.

NLpep WT, NLpep78, NLpep79 or NLpep 80 (synthetic) were diluted to 10 nMin PBS+0.1% Prionex, and 25 μl mixed with 25 uL of each of the NLpolycell lysate. The samples were shaken for 10 min at RT, and then 50 uLNanoGlo+100 uM Fz added and incubated for 5 min at RT. Luminescence wasmeasured on a GloMax luminometer with 0.5 s integration. FIG. 102illustrates the improvement in luminescence from HEK293-expressed NLpolyover the course of the evolution process, an overall ˜10⁴ improvement(from NLpolyWT:NLpepWT to NLpoly11S:NLpep80).

Example 61 Improved Binding Affinity Throughout Evolution

NLpoly WT, 5A2, 5P, 8S or 11S (E. coli clarified lysates) were dilutedinto PBS+0.1% Prionex as follows: WT 1:10⁴, 5 A2 1:105; 5P 1:10⁶; 8S1:10⁷; and 11S 1:10⁷. NLpepWT, NLpep78, NLpep79 or NLpep80 (synthetic)were serially into PBS+0.1% Prionex to 4× concentration. 25 uL NLpolyand 25 uL NLpep were mixed and incubated for 10 min at RT. 50 uLNanoGlo+100 uM Fz was added and incubated for 5 min at RT. Luminescencewas measured on a GloMax Multi+ with 0.5 sec integration. Kd wasdetermined using Graphpad Prism, One Site-Specific Binding, Best-fitvalues. FIG. 103 illustrates a 10⁴ fold improved affinity (startingaffinity: NLpolyWT:NLpepWT, Kd˜10 uM) of K_(d)<1 nM (NLpoly11S:NLpep86or NLpoly11S:NLpep80) of the variants tested over wild-type.

Example 62 NLpoly Luminescence

Single NLpoly variant colonies were inoculated with 200 uL minimal mediaand grown for 20 hrs at 37° C. on a shaker. 10 uL of the overnightculture were diluted into 190 uL fresh minimal media and grown for 20hrs at 37° C. on a shaker. 10 uL of this overnight culture was thendiluted into 190 uL auto-induction media (previously described) andgrown for 18 hrs at 25° C. on a shaker. 10 uL of this expression culturewas mixed with 40 uL of assay lysis buffer (previously described)without NLpep or NLpep78-HT (1:3, 860 dilution) or NLpep79-HT (1:10,000dilution) and shaken for 10 min at RT. 50 uL of NanoGlo+Fz was added andagain shake for 10 min at RT. Luminescence was measured on GloMax®luminometer with 0.5 sec integration. FIGS. 105-107 illustrate theluminescence of various NLpolys in the absence of NLpep.

Example 63 Solubility of NLpoly Variants

A single NLpoly variant colony (SEE FIG. 143) was inoculated into 5 mLLB culture and incubated at 37° C. overnight with shaking. The overnightculture was diluted 1:100 into fresh LB and incubated at 37° C. for 3hrs with shaking. Rhamnose was added to the cultures to 0.2% andincubated 25° C. overnight with shaking. 900 μl of these overnightcultures were mixed with 100 uL 10× FastBreak Lysis Buffer (PromegaCorporation) and incubated for 15 min at RT. A 75 uL aliquot (total) wasremoved from each culture and saved for analysis. The remaining culturefrom each sample were centrifuged at 14,000×rpm in a benchtopmicrocentrifuge at 4° C. for 15 min. A 75 uL aliquot of supernatant(soluble) was removed from each sample and saved for analysis. 25 uL of4×SDS buffer was added to the saved aliquots and incubated at 95° C. for5 min. 5 ul of each sample was loaded onto a 4-20% Tris-Glycine SDS geland run at ˜190V for ˜50 min. The gel was stained with SimplyBlue SafeStain and imaged on a LAS4000.

FIG. 143 shows a protein gel of total lysates and the soluble fractionof the same lysate for the NLpoly variants.

Example 64 Dissociation Constants

NLpoly variant lysate (SEE FIG. 144; prepared as described previously)was diluted 1:10 into PBS+0.1% Prionex. 4× concentrations of NLpep78(synthetic NLpep78) were made in PBS+0.1% Prionex. 20 uL NLpoly variantlysate and 20 uL NLpep were mixed and shaken for 10 min at RT. 40 uLNanoGlo/Fz was added and shaken for 10 min at RT. Luminescence wasmeasured on a GloMax® luminometer with 0.5 s integration. Kd determinedusing Graphpad Prism, One site-specific binding, best-fit values. FIG.144 illustrates dissociation constants of NLpep78 with various NLpolys.

Example 65 Comparison of Luminescence Generated by Cells ExpressingDifferent Combinations of FRB and FKBP Fused to NLpoly5P and NLpep80/87

HEK293T cells (400,000) were reverse-transfected with 1 μg pF4A Ag FKBPor 1 μg pF4A Ag FRB vectors expressing N- or C-terminal fusions ofNLpoly5P and/or NLpep80/87 using FuGENE HD at a DNA-to-FuGENE HD ratioof 1:4. 24-hours post transfection, cells were trypsinized and re-platedin opaque 96-well assay plates at a density of 10,000 cells per well.24-hours after plating, cells were washed with PBS and then incubatedwith or without 20 nM rapamycin for 15, 60 or 120 min in phenol red-freeOptiMEMI. 10 μM furimazine substrate with or without 20 nM rapamycin inOptiMEM was added directly to each well and incubated at roomtemperature for 5 min. Luminescence was then measured on a GloMax Multiwith 0.5 s integration time. FIGS. 108 (15 min induction), 109 (60 mininduction) and 110 (120 min induction) illustrate a general increase ininduction over time, with NLpoly5P and NLpep80 combinations generatingthe most luminescence. Individual components contribute minimally tosignal.

Example 66 Comparison of Luminescence Generated by Cells ExpressingDifferent Combinations of FRB and FKBP Fused to NLpoly5P and NLpep80/87

Although similar to Example 65, this example tested all 8 possiblecombinations of FRB and FKBP fused to NLpoly/NLpep as well as used lesstotal DNA. HEK293T cells (400,000) were reverse-transfected with a totalof 0.001 μg pF4A Ag FRB-NLpoly5P and 0.001 μg pF4A AgFKBP-NLpep80/NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8.pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque96-well assay plates and incubated an additional 24 hours. Cells werewashed with PBS and then incubated in phenol red-free OptiMEMI with 0 or50 nM rapamycin for 2 h. 10 μM furimazine substrate (final concentrationon cells) with 0 or 50 nM rapamycin in OptiMEM was added directly toeach well and incubated at room temperature for 5 min. Luminescence wasthen measured on a GloMax Multi with 0.5 s integration time. FIG. 111illustrates that NLpep80 combinations generated the highest luminescenceand that all configurations respond to rapamycin treatment.

Example 67 Comparison of Luminescence Generated by FRB or FKBP FusionsExpressed in the Absence of Binding Partner

HEK293T cells (400,000) were reverse-transfected with a total of 0.001μg pF4A Ag FRB-NLpoly5P or pF4A Ag FKBP-NLpep80/NLpep87 using FuGENE HDat a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bringtotal DNA in each transfection to 1 μg. 24-hours post-transfection,10,000 cells were re-plated in opaque 96-well assay plates and incubatedan additional 24 hours. Cells were washed with PBS and then incubated inphenol red-free OptiMEMI with 0 or 50 nM rapamycin for 2 h. 10 μMfurimazine substrate (final concentration on cells) with 0 or 50 nMrapamycin in OptiMEM was added directly to each well and incubated atroom temperature for 5 min. Luminescence was then measured on a GloMaxMulti with 0.5 s integration time. FIG. 112 illustrates that theindividual components generate a low basal level of luminescence that isnot responsive to rapamycin treatment.

Example 68 Comparison of Luminescence Generated by Cells Transfectedwith Varying Amounts of FRB-NLpoly5P and FKBP-NLpep80/87 DNA

HEK293T (400,000) cells were reverse-transfected with a total of 2, 0.2,0.02, or 0.002 μg pF4A Ag FRB-NLpoly5P and pF4A Ag FKBP-NLpep80 usingFuGENE HD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was addedto bring total DNA in each transfection to 2 μg. 24-hourspost-transfection, 10,000 cells were re-plated in opaque 96-well assayplates and incubated an additional 24 hours. Cells were washed with PBSand then incubated in phenol red-free OptiMEMI with or without 20 nMrapamycin for 2 h. 10 μM furimazine substrate (final concentration oncells) with or without 20 nM rapamycin in OptiMEM was added directly toeach well and incubated at room temperature for 5 min. Luminescence wasthen measured on a GloMax Multi with 0.5 s integration time. FIG. 113illustrates that transfection with less DNA decreases overallluminescence but increases fold induction.

Example 69 Comparison of Luminescence Generated by Cells Transfectedwith Varying Amounts of FRB-NLpoly5P or FKBP-NLpep80/87 DNA in theAbsence of Binding Partner

HEK293T cells (400,000) were reverse-transfected with a total of 2, 0.2,0.02, or 0.002 μg pF4A Ag FRB-NLpoly5P or pF4A Ag FKBP-NLpep80 usingFuGENE HD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was addedto bring total DNA in each transfection to 2 μg. 24-hourspost-transfection, 10,000 cells were replated in opaque 96-well assayplates and incubated an additional 24 hours. Cells were washed with PBSand then incubated in phenol red-free OptiMEMI with or without 20 nMrapamycin for 2 h. 10 μM furimazine substrate (final concentration oncells) with or without 20 nM rapamycin in OptiMEM was added directly toeach well and incubated at room temperature for 5 min. Luminescence wasthen measured on a GloMax Multi with 0.5 s integration time. FIG. 114illustrates that lower DNA levels do not change overall luminescence ofcells transfected with individual components.

Example 70 Comparison of Luminescence Generated by Cells Transfectedwith Varying Amounts of FRB-NLpoly5P and FKBP-NLpep80/87 DNA

HEK293T cells (400,000) were reverse-transfected with a total of 0.2,0.02, 0.002, or 0.0002 μg pF4A Ag FRB-NLpoly5P and pF4A AgFKBP-NLpep80/NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4.pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 2μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque96-well assay plates and incubated an additional 24 hours. Cells werewashed with PBS and then incubated in phenol red-free OptiMEMI with orwithout 50 nM rapamycin for 2 h. 10 μM furimazine substrate (finalconcentration on cells) with or without 50 nM rapamycin in OptiMEM wasadded directly to each well and incubated at room temperature for 5 min.Luminescence was then measured on a GloMax Multi with 0.5 s integrationtime. FIG. 115 illustrates that luminescence above background, asdetermined in Examples 69 and 71, and rapamycin induction can beachieved with DNA levels down to 2.5 pg.

Example 71 Comparison of Luminescence Generated by Cells Transfectedwith Varying Amounts of FRB-NLpoly5P or FKBP-NLpep80/87 DNA in theAbsence of Binding Partner

HEK293T cells (400,000) were reverse-transfected with a total of 0.2,0.02, 0.002, or 0.0002 μg pF4A Ag FRB-NLpoly5P or pF4A AgFKBP-NLpep80/NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4.pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 2μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque96-well assay plates and incubated an additional 24 hours. Cells werewashed with PBS and then incubated in phenol red-free OptiMEMI with orwithout 50 nM rapamycin for 2 h. 10 μM furimazine substrate (finalconcentration on cells) with or without 50 nM rapamycin in OptiMEM wasadded directly to each well and incubated at room temperature for 5 min.Luminescence was then measured on a GloMax Multi with 0.5 s integrationtime. FIG. 116 illustrates no significant change in luminescencegenerated by individual components when less DNA was used.

Example 72 Comparison of Luminescence Generated by Cells Transfectedwith Varying Amounts of FRB-NLpoly5P and FKBP-NLpep80 or FKBP-NLpep87DNA after Treatment with Rapamycin for Different Lengths of Time

HEK293T cells (400,000) were reverse-transfected with a total of 2, 0.2,0.02, or 0.002 μg pF4A Ag FRB-NLpoly5P and pF4A Ag FKBP-NLpep80 orFKBP-NLpep87 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 4.pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 2μg. 24-hours post-transfection, 10,000 cells were re-plated in opaque96-well assay plates and incubated an additional 24 hours. Cells werewashed with PBS and then incubated in phenol red-free OptiMEMI with orwithout 20 nM rapamycin for 5/15/30/60/120 min. 10 μM furimazinesubstrate (final concentration on cells) with or without 20 nM rapamycinin OptiMEM was added directly to each well and incubated at roomtemperature for 5 min. Luminescence was then measured on a GloMax Multiwith 0.5 s integration time. FIGS. 117 and 118 illustrates a decline inluminescence with less DNA and an increase in rapamycin induction overtime.

Example 73 Comparison of Luminescence Generated by Cells ExpressingDifferent Combinations of FRB-NLpoly5P or FRB-NLpoly5A2 withFKBP-NLpep80/87/95/96/97

In this example, the assay was performed in both a two-day and three-dayformat. For the 2 day assay, 20,000 HEK293T cells werereverse-transfected in opaque 96-well assay plates with a total of 0.1ng pF4A Ag FRB-NLpoly5P or FRB-NLpoly5A2 and pF4A AgFKBP-NLpep80/87/95/96/97 using FuGENE HD at a DNA-to-FuGENE ratio of 1to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfectionto 1 ug. 24 hours-post transfection, cells were washed with PBS and thenincubated in phenol red-free OptiMEMI with or without 50 nM rapamycinfor 2 h. 10 μM furimazine substrate (final concentration on cells) withor without 50 nM rapamycin in OptiMEMI was added directly to each welland incubated at room temperature for 5 min. Luminescence was thenmeasured on a GloMax Multi with 0.5 s integration time.

For 3 day assay, 400,000 HEK293T cells were reverse-transfected with atotal of 0.002 μg pF4A Ag FRB-NLpoly5P and pF4A AgFKBP-NLpep80/87/95/96/97 using FuGENE HD at a DNA-to-FuGENE ratio of 1to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfectionto 1 ug. 24-hours post-transfection, 10,000 cells were re-plated inopaque 96-well assay plates and incubated an additional 24 hours. Cellswere washed with PBS and then incubated in phenol red-free OptiMEMI withor without 50 nM rapamycin for 2 h. 10 μM furimazine substrate (finalconcentration on cells) with or without 50 nM rapamycin in OptiMEMI wasadded directly to each well and incubated at room temperature for 5 min.Luminescence was then measured on a GloMax Multi with 0.5 s integrationtime. FIGS. 119 and 120 illustrate similar levels of luminescence inboth the 2 day and 3 day assays. Assays performed with NLpoly5A2 showedgreater rapamycin induction relative to NLpoly5P, and assays performedwith NLpoly5A2 and NLpep96 showed greatest rapamycin induction of alltested combinations.

Example 73 Comparison of Luminescence Generated by Cells ExpressingDifferent Combinations of FRB-NLpoly5A2 or FRB-NLpoly11S withFKBP-NLpep101/104/105/106/107/108/109/110

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assayplates with a total of 0.1 ng pF4A Ag FRB-NLpoly5A2/11S and pF4A AgFKBP-NLpep101/104/105/106/107/108/109/110 using FuGENE HD at aDNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring totalDNA in each transfection to 1 μg. 24 hours-post transfection, cells werewashed with PBS and then incubated in phenol red-free OptiMEMI with orwithout 50 nM rapamycin for 2 h. 10 μM furimazine substrate (finalconcentration on cells) with or without 50 nM rapamycin in OptiMEMI wasadded directly to each well and incubated at room temperature for 5 min.Luminescence was then measured on a GloMax Multi with 0.5 s integrationtime. FIG. 121 illustrates that, of tested combinations, NLpoly11S withNLpep101 showed the greatest rapamycin induction and one of thestrongest rapamycin-specific luminescent signals.

Example 74 Comparison of Luminescence Generated by Cells Transfectedwith Different Combinations of FRB-NLpoly5A2 or FRB-NLpoly11S withFKBP-NLpep87/96/98/99/100/101/102/103

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assayplates with a total of 0.1 ng pF4A Ag FRB-NLpoly5A2/11S and pF4A AgFKBP-NLpep87/96/98/99/100/101/102/103 using FuGENE HD at a DNA-to-FuGENEratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in eachtransfection to 1 μg. 24 hours-post transfection, cells were washed withPBS and then incubated in phenol red-free OptiMEMI with or without 50 nMrapamycin for 2 h. 10 μM furimazine substrate (final concentration oncells) with or without 50 nM rapamycin in OptiMEMI was added directly toeach well and incubated at room temperature for 5 min. Luminescence wasthen measured on a GloMax Multi with 0.5 s integration time. FIG. 122illustrates that the NLpoly11S and NLpep101 combination produces thehighest induction while maintaining high levels of specificluminescence.

Example 75 Comparison of Luminescence Generated by Cells Transfectedwith Different Levels of FRB-NLpoly11S and FKBP-NLpep87/101/102/107 DNA

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assayplates with a total of 0.01, 0.1, 1, or 10 ng pF4A Ag FRB-NLpoly11S andpF4A Ag FKBP-NLpep87/101/102/107 using FuGENE HD at a DNA-to-FuGENEratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in eachtransfection to 1 ug. 24 hours-post transfection, cells were washed withPBS and then incubated in phenol red-free OptiMEMI with or without 50 nMrapamycin for 1.5 h. 10 μM furimazine substrate (final concentration oncells) with or without 50 nM rapamycin in OptiMEMI was added directly toeach well and incubated at room temperature for 5 min. Luminescence wasthen measured on a GloMax Multi with 0.5 s integration time. FIG. 123illustrates NLpoly11S with NLpep101 produces the overall lowestluminescence in untreated samples at all tested DNA levels, and thecombination maintains relatively high levels of luminescence inrapamycin-treated samples.

Example 76 Comparison of Luminescence Generated by Cells Transfectedwith Different Levels of FRB-NLpoly5A2 and FKBP-NLpep87/101/102/107 DNA

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assayplates with a total of 0.01, 0.1, 1, or 10 ng pF4A Ag FRB-NLpoly5A2 andpF4A Ag FKBP-NLpep87/101/102/107 using FuGENE HD at a DNA-to-FuGENEratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in eachtransfection to 1 μg. 24 hours-post transfection, cells were washed withPBS and then incubated in phenol red-free OptiMEMI with or without 50 nMrapamycin for 1.5 h. 10 μM furimazine substrate (final concentration oncells) with or without 50 nM rapamycin in OptiMEMI was added directly toeach well and incubated at room temperature for 5 min. Luminescence wasthen measured on a GloMax Multi with 0.5 s integration time. FIG. 124illustrates that NLpoly5A2 generates higher luminescence in untreatedsamples than NLpoly11S shown in example 75.

Example 77 Rapamycin Dose Response Curve Showing Luminescence of CellsExpressing FRB-NLpoly5P and FKBP-NLpep80/87 DNA

HEK293T cells (400,000) were reverse-transfected with a total of 0.001μg pF4A Ag FRB-NLpoly5P and 0.001 μg pF4A Ag FKBP-NLpep80/NLpep87 usingFuGENE HD at a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was addedto bring total DNA in each transfection to 1 μg. 24-hourspost-transfection, 10,000 cells were re-plated in opaque 96-well assayplates and incubated an additional 24 hours. Cells were washed with PBSand then incubated in phenol red-free OptiMEMI with 0 to 500 nMrapamycin for 2 h. 10 μM furimazine substrate (final concentration oncells) with 0 to 500 nM rapamycin in OptiMEM was added directly to eachwell and incubated at room temperature for 5 min. Luminescence was thenmeasured on a GloMax Multi with 0.5 s integration time. Kd wascalculated with GraphPad Prism version 5.00 for Windows. FIG. 125illustrates a rapamycin-specific increase in luminescence.

Example 78 Rapamycin Dose Response Curve Showing Luminescence of CellsExpressing FRB-NLpoly5A2 and FKBP-NLpep87/101 DNA

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assayplates with a total of 0.1 ng pF4A Ag FRB-NLpoly5A2/11S and pF4A AgFKBP-NLpep87/101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8.pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1ug. 24 hours-post transfection, cells were washed with PBS and thenincubated in phenol red-free OptiMEMI with 0 to 1 μM rapamycin for 1.5h. 10 μM furimazine substrate (final concentration on cells) with 0 to 1μM rapamycin in OptiMEMI was added directly to each well and incubatedat room temperature for 5 min. Luminescence was then measured on aGloMax Multi with 0.5 s integration time. FIG. 126 illustrates asigmoidal dose response to rapamycin with NLpoly5A2/NLpep101 andNLpoly11S/NLpep101 combinations. While combinations with NLpep87 show anincrease in luminescence with rapamycin, the collected data pointsdeviate more from the sigmoidal curve.

Example 79 Comparison of Luminescence Generated by Cells ExpressingFRB-11S and FKBP-101 and Treated with Substrate PBI-4377 or Furimazine

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assayplates with a total of 0.1/1/10 ng pF4A Ag FRB-NLpoly11S and pF4A AgFKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8.pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1ug. 24 hours-post transfection, cells were washed with PBS and thenincubated in phenol red-free OptiMEMI with 0 or 50 nM rapamycin for 1.5h. 10 μM furimazine or PBI-4377 substrate (final concentration on cells)with 0 to 50 nM rapamycin in OptiMEMI was added directly to each welland incubated at room temperature for 5 min. Luminescence was thenmeasured on a GloMax Multi with 0.5 s integration time. FIG. 127illustrates a decrease in luminescence and fold induction with thePBI-4377 substrate compared to the furimazine substrate.

Example 80 Time Course of Cells Expressing FRB-NLpoly11S/5A2 andFKBP-NLpep87/101 Conducted in the Presence or Absence of Rapamycin

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assayplates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S/5A2 and pF4A AgFKBP-NLpep87/101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8.pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1μg. 24 hours-post transfection, cells were washed with PBS and thenphenol red-free OptiMEMI with 0 or 50 nM rapamycin and 10 μM furimazinewas added either manually or via instrument injection. Luminescence wasimmediately measured on a GloMax Multi with 0.5 s integration time.FIGS. 128 and 129 illustrate that, of all combinations tested, NLpoly11Swith NLpep101 has the lowest luminescence at time 0, hits a luminescentplateau faster and has the largest dynamic range.

Example 81 Luminescence Generated by FRB-NLpoly11S and FKBP-NLpep101 asMeasured on Two Different Instruments

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assayplates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S and pF4A AgFKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8.pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1μg. 24 hours-post transfection, cells were washed with PBS and thenphenol red-free OptiMEMI with 0 or 50 nM rapamycin was added for 20 min.10 μM furimazine (final concentration on cells) in OptiMEMI with 0 or 50nM rapamycin was added and incubated for an additional 5 min.Luminescence was immediately measured on a GloMax Multi with 0.5 sintegration time and on the Varioskan Flash with 450 nM band passfilter. FIG. 130 illustrates that the rapamycin-specific induction ofFRB-NLpoly11S and FKBP-NLpep101 can be measured on differentinstruments.

Example 82 Images Showing Luminescence of Cells Expressing FRB-NLpoly11Sand FKBP-NLpep101 at Various Times after Treatment with Rapamycin

HeLa cells (500,000) were reverse transfected with 1 μg pF4 AgFRB-NLpoly11S and 1 μg pF4 Ag FKBP-NLpep101 using FuGENE HD at a DNA toFuGENE ratio of 1 to 4. Cells were transfected in 35 mm glass bottomculture dishes (MatTek #p35gc-1.5-14-C). 24 hours post-transfection,cells were washed with PBS and then incubate with 10 μM furimazine inOptiMEM for 5 min. 50 nM rapamycin in OptiMEMI was added to cells andluminescent images were acquired with LV200 at 10 s intervals for atotal of 20 min. Instrument was at 37° C., objective was 60×, gain was200 and exposure was 600 ms. FIG. 131 illustrates that imaging candetect an increase in cellular luminescence in cells expressingFRB-NLpoly11S and FKBP-NLpep101 following rapamycin treatment.

Example 83 Quantitation of the Signal Generated by Individual CellsExpressing FRB-NLpoly11S and FKBP-NLpep101 at Various Times afterTreatment with Rapamycin

HeLa cells (500,000) were reverse transfected with 1 μg pF4 AgFRB-NLpoly11S and 1 μg pF4 Ag FKBP-NLpep101 using FuGENE HD at a DNA toFuGENE ratio of 1 to 4. Cells were transfected in 35 mm glass bottomculture dishes (MatTek #p35gc-1.5-14-C). 24 hours post-transfection,cells were washed with PBS and then incubate with 10 μM furimazine inOptiMEM for 5 min. 50 nM rapamycin in OptiMEMI was added to cells, andluminescent images were acquired with LV200 at 10 s intervals for atotal of 20 min. Instrument was at 37° C., objective was 60×, gain was200, and exposure was 600 ms. The signal intensity of every cell in thefield of view was analyzed with Image J software over the entire timeperiod. FIG. 132 illustrates that signal generated by individual cellscan be measured and that the increase in signal by each cell parallelsthe increase observed in the 96-well plate assay shown in FIGS. 128 and129.

Example 84 Comparison of Luminescence in Different Cell Lines ExpressingFRB-NLpoly11S and FKBP-NLpep101

HEK293T, HeLa, or U2-OS cells (20,000) were reverse-transfected inopaque 96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly11Sand pF4A Ag FKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1to 8. pGEM-3Zf(+) DNA was added to bring total DNA in each transfectionto 1 μg. 24 hours-post transfection, cells were washed with PBS and thenphenol red-free OptiMEMI with 0 or 50 nM rapamycin was added for 20 min.10 μM furimazine (final concentration on cells) in OptiMEMI with 0 or 50nM rapamycin was added and incubated for an additional 5 min.Luminescence was immediately measured on a GloMax Multi with 0.5 sintegration time. FIG. 133 illustrates similar levels of luminescencegenerated in the absence and presence of rapamycin in three differentcells lines transfected with FRB-NLpoly11S and FKBP-NLpep101.

Example 85 Comparison of Luminescence Generated by Cells ExpressingFRB-NLpoly11S and FKBP-NLpep101 after Treatment with the RapamycinCompetitive Inhibitor FK506

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assayplates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S and pF4A AgFKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8.pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1μg. 24 hours-post transfection, cells were washed with PBS and thenphenol red-free OptiMEMI with 0 or 20 nM rapamycin was added for 20 min.FK506 inhibitor in OptiMEM was added to cell at final concentration of 5μM and incubated for 3 or 5 hours. Furimazine in OptiMEM was added tocells for a final concentration of 10 μM on cells. Luminescence wasimmediately measured on a GloMax Multi with 0.5 s integration time. FIG.134 illustrates a decrease in rapamycin-induced luminescence aftertreatment with the competitive inhibitor FK506.

Example 86 Luminescence Generated by Cells Expressing FRB-NLpoly11S andFKBP-NLpep101 after Treatment with the Rapamycin Competitive InhibitorFK506

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assayplates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S and pF4A AgFKBP-NLpep101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8.pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1μg. 24 hours-post transfection, cells were washed with PBS and thenphenol red-free OptiMEMI with 0 or 20 nM rapamycin was added for 2.5hours. FK506 inhibitor in OptiMEM was added to cell via injector atfinal concentration of 0, 1 or 10 μM in OptiMEM with 10 μM. Luminescencewas measured every 10 min for 4 hours on a GloMax Multi set to 37° C.with 0.5 s integration time. FIG. 135 illustrates that by 200 s, FK506inhibitor can reduce luminescence close to levels of untreated cells.

Example 87 Luminescence Generated by Cells Transfected with DifferentCombinations of V2R-NLpoly5A2 or V2R-NLpoly11S with NLpep87/101-ARRB2 inthe Presence or Absence of the V2R Agonist AVP

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assayplates with a total of 0.1, 1, or 10 ng pF4A Ag V2R-NLpoly11S and pF4AAg ARRB2-NLpep87/101 using FuGENE HD at a DNA-to-FuGENE ratio of 1 to 8.pGEM-3Zf(+) DNA was added to bring total DNA in each transfection to 1μg. 24 hours-post transfection, cells were washed with PBS and thenphenol red-free OptiMEMI with 0 or 1 μM AVP and 10 μM furimazine wasadded for 25 min. Luminescence was then measured on a GloMax Multi with0.5 s integration time.

FIG. 136 illustrates that V2R-NLpoly11S with NLpep101 generates thegreatest AVP-specific increase in luminescence. Combinations withNLpep87 show no significant response to AVP.

Example 88 Time Course Showing Luminescence Generated by CellsTransfected with V2R-NLpoly5A2 or V2R-NLpoly11S and NLpep87/101-ARRB2after Treatment with AVP

HEK293T cells (20,000) were reverse-transfected in opaque 96-well assayplates with a total of 0.1 or 1 ng pF4A Ag V2R-NLpoly11S or 1 ng pF4A AgV2R-NLpoly5A2 and pF4A Ag ARRB2-NLpep87/101 using FuGENE HD at aDNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring totalDNA in each transfection to 1 μg. 24 hours-post transfection, cells werewashed with PBS and then phenol red-free OptiMEMI with 0 or 1 μM AVP and10 μM furimazine was added either manually (FIG. 137) or via instrumentinjection (FIG. 138). Luminescence was then measured on a GloMax Multievery 5 min for 25 min with 0.5 s integration time at room temperature(FIGS. 137 and 138) or 37° C. (FIG. 139). FIGS. 137 and 138 illustrate atime-dependent increase in AVP-induced luminescence for V2R-NLpoly11Swith NLpep101-ARRB2 that begins to peak at 600 s. Combinations withV2R-NLpoly5A2 and NLpep87 do not show a significant increase inluminescence over time. FIG. 139 illustrates that at 37° C. allNLpoly11S and NLpep101 combinations tested show a time-dependentincrease in AVP-induced luminescence that levels out around 200 s.

Example 89 Comparison of Luminescence in Different Cell Lines ExpressingV2R-NLpoly11S and NLpep101-ARRB2

HEK293T, HeLa, or U2-OS cells (20,000) were reverse-transfected inopaque 96-well assay plates with a total of 1 ng pF4A Ag V2R-NLpoly11Sand pF4A Ag ARRB2-NLpep87/101 using FuGENE HD at a DNA-to-FuGENE ratioof 1 to 8. pGEM-3Zf(+) DNA was added to bring total DNA in eachtransfection to 1 μg. 24 hours-post transfection, cells were washed withPBS and then phenol red-free OptiMEMI with 0 or 1 μM AVP was added for20 min. Furimazine in OptiMEM was then added to a final concentration of10 μM on cells, and luminescence was measured on a GloMax Multi with 0.5s integration time.

FIG. 140 illustrates similar luminescence levels in three different celllines expressing V2R-NLpoly11S and NLpep101-ARRB2 in the presence andabsence of AVP.

Example 90 Luminescence of Cells Expressing V2R-NLpoly11S andNLpep101-ARRB2 at Various Times after Treatment with AVP

HeLa cells (500,000) were reverse transfected with 1 μg pF4 AgV2R-NLpoly11S and 1 ug pF4 Ag ARRB2-NLpep101 using FuGENE HD at a DNA toFuGENE ratio of 1 to 4. Cells were transfected in 35 mm glass bottomculture dishes (MatTek #p35gc-1.5-14-C). 24 hours post-transfection,cells were washed with PBS and then incubate with 10 μM furimazine inOptiMEM for 5 min. 1 μM AVP in OptiMEMI was added to cells, andluminescent images were acquired with LV200 at 15 s intervals for atotal of 30 min. Instrument was at 37° C., objective was 60× or 150×,gain was 600, and exposure was is or 2 s. FIGS. 141 and 142 illustratethat imaging can detect the increase in luminescence and formation ofpunctate in individual cells after treatment with AVP.

Example 91 Dissociation Constants for NLpeps

NLpoly 5P E. coli clarified lysate (prepared as described previously)was diluted 1:1,000 into PBS+0.1% Prionex. 4× concentrations ofNLpep78-HT (E. coli clarified lysate prepared as described previously)were made in PBS+0.1% Prionex. 20 uL NLpoly 5P and 20 uL NLpep78 weremixed and shaken for 10 min at RT. 40 uL NanoGlo/Fz was added and shakenfor 10 min at RT. Luminescence was measured on GloMax luminometer with0.5 s integration. Kd was determined using Graphpad Prism, Onesite-specific binding, best-fit values. FIG. 80 compares thedissociation constants for an NLpep consisting of either 1 or 2 repeatunits of NLpep78.

Example 92 Affinity Between NLpoly 5A2 and NLpep86

NLpoly 5A2 lysate (prepared as described previously after transfectingCHO cells) was diluted 1:10 into PBS+0.1% Prionex. 4× concentrations ofNLpep86 (synthetic NLpep) were made in PBS+0.1% Prionex. 20 uL NLpolyand 20 uL NLpep were mixed and shaken for 10 min at RT. 40 uL NanoGlo/Fzwas added and shaken for 10 min at RT. Luminescence was measured onGloMax luminometer with 0.5 s integration. Kd was determined usingGraphpad Prism, One site-specific binding, best-fit values. FIG. 81illustrates the affinity between NLpoly 5A2 and NLpep86.

Example 93 Luminescence of NLpoly Variants

A single colony of various NLpolys were inoculated individually into 200uL minimal media and grown for 20 hrs at 37° C. on shaker. 10 uL ofovernight culture was diluted into 190 uL fresh minimal media and grownfor 20 hrs at 37° C. on shaker. 10 uL of this overnight culture wasdiluted into 190 uL auto-induction media (previously described) and growfor 18 hrs at 25° C. on shaker. 10 uL of the expression culture wasmixed with 40 uL of assay lysis buffer (previously described) withoutNLpep or with NLpep78-HT (1:3, 860 dilution) or NLpep79-HT (1:10,000dilution). The mixtures were shaken for 10 min at RT, 50 uL NanoGlo+Fzadded and shaken again for 10 min at RT. Luminescence was measured on aGloMax luminometer with 0.5 sec integration. FIG. 82 demonstrates theluminescence from NLpoly variants without an NLpep or with NLpep78 orNLpep79. The results show that the NLpoly variant 11S (12S-51) hasimproved luminescence over the other variants.

Example 94 Dissociation Constants and Vmax Values for NLpolys with 96Variants of NLpeps

NLpeps were synthesized in array format by New England Peptide (peptidesblocked at N-terminus by acetylation and at C-terminus by amidation;peptides in arrays were synthesized at ˜1 mg scale) (Table 6). Eachpeptide was lyophilized in 3 separate plates. Each well from 1 of the 3plates of peptides was dissolved in 100 uL nanopure water, and the A260measured and used to calculate the concentration using the extinctioncoefficient of each peptide. The concentration was then adjusted basedon the purity of the peptide, and nanopure water was added to give afinal concentration of 750 uM.

Peptides were diluted to 12.66 uM (4×) in PBS+0.1% Prionex and thendiluted serially 7 times (8 concentrations total) in 0.5 log steps(3.162 fold dilution). NLpolys 5P, 8S, 5A2 or 11S were diluted intoPBS+0.1% Prionex as follows: 5P 1:2,000; 8S 1:10,000; 11S 1:150,000, 5A21:1,000. 25 uL each NLpep+25 uL each NLpoly were mixed and incubated for30 min at RT. 50 uL NanoGlo+100 uM Fz was added and incubated for 30 minat RT. Luminescence was measure on a GloMax Multi+ with 0.5 secintegration. Kd/Vmax were determined using Graphpad Prism, Onesite-specific binding, best-fit values. FIGS. 83-90 illustrate thedissociation constant and Vmax values from NLpolys with the 96 variantNLpeps. The results indicate specific mutations in the NLpeps thatexhibit lower binding affinity without loss in Vmax.

TABLE 6  Peptide Array 1 Sequence SEQ ID NO. array1.1 VTGWRLCERIL 2366array1.2 VSGWRLFKKIS 2367 array1.3 VTGYRLFKKIS 2368 array1.4 ISGWRLFKKIS2369 array1.5 ASGWRLFKKIS 2370 array1.6 GSGWRLFKKIS 2371 array1.7KSGWRLFKKIS 2372 array1.8 LSGWRLFKKIS 2373 array1.9 QSGWRLFKKIS 2374array1.10 SSGWRLFKKIS 2375 array1.11 TSGWRLFKKIS 2376 array1.12VVGWRLFKKIS 2377 array1.13 VKGWRLFKKIS 2378 array1.14 VIGWRLFKKIS 2379array1.15 VEGWRLFKKIS 2380 array1.16 VAGWRLFKKIS 2381 array1.17VQGWRLFKKIS 2382 array1.18 VHGWRLFKKIS 2383 array1.19 VSAWRLFKKIS 2384array1.20 VSSWRLFKKIS 2385 array1.21 VSGFRLFKKIS 2386 array1.22VSGWKLFKKIS 2387 array1.23 VSGWQLFKKIS 2388 array1.24 VSGWELFKKIS 2389array1.25 VSGWALFKKIS 2390 array1.26 VSGWRIFKKIS 2391 array1.27VSGWRVFKKIS 2392 array1.28 VSGWRTFKKIS 2393 array1.29 VSGWRYFKKIS 2394array1.30 VSGWRKFKKIS 2395 array1.31 VSGWRFFKKIS 2396 array1.32VSGWRLAKKIS 2397 array1.33 VSGWRLDKKIS 2398 array1.34 VSGWRLEKKIS 2399array1.35 VSGWRLGKKIS 2400 array1.36 VSGWRLHKKIS 2401 array1.37VSGWRLIKKIS 2402 array1.38 VSGWRLKKKIS 2403 array1.39 VSGWRLLKKIS 2404array1.40 VSGWRLMKKIS 2405 array1.41 VSGWRLNKKIS 2406 array1.42VSGWRLQKKIS 2407 array1.43 VSGWRLRKKIS 2408 array1.44 VSGWRLSKKIS 2409array1.45 VSGWRLTKKIS 2410 array1.46 VSGWRLVKKIS 2411 array1.47VSGWRLWKKIS 2412 array1.48 VSGWRLYKKIS 2413 array1.49 VSGWRLFEKIS 2414array1.50 VSGWRLFVKIS 2415 array1.51 VSGWRLFSKIS 2416 array1.52VSGWRLFRKIS 2417 array1.53 VSGWRLFTKIS 2418 array1.54 VSGWRLFNKIS 2419array1.55 VSGWRLFQKIS 2420 array1.56 VSGWRLFKRIS 2421 array1.57VSGWRLFKQIS 2422 array1.58 VSGWRLFKEIS 2423 array1.59 VSGWRLFKAIS 2424array1.60 VSGWRLFKKVS 2425 array1.61 VSGWRLFKKLS 2426 array1.62VSGWRLFKKAS 2427 array1.63 VSGWRLFKKFS 2428 array1.64 VSGWRLFKKES 2429array1.65 VSGWRLFKKTS 2430 array1.66 VSGWRLFKKIL 2431 array1.67VSGWRLFKKIA 2432 array1.68 VSGWRLFKKIE 2433 array1.69 VSGWRLFKKIV 2434array1.70 VSGWRLFKKIG 2435 array1.71 VSGWRLFKKIH 2436 array1.72VSGWRLFKKIT 2437 array1.73 VVGYRLFKKIS 2438 array1.74 VKGYRLFKKIS 2439array1.75 VIGYRLFKKIS 2440 array1.76 VEGYRLFKKIS 2441 array1.77VAGYRLFKKIS 2442 array1.78 VQGYRLFKKIS 2443 array1.79 VHGYRLFKKIS 2444array1.80 VTAYRLFKKIS 2445 array1.81 VTSYRLFKKIS 2446 array1.82VTGYRIFKKIS 2447 array1.83 VTGYRVFKKIS 2448 array1.84 VTGYRTFKKIS 2449array1.85 VTGYRYFKKIS 2450 array1.86 VTGYRKFKKIS 2451 array1.87VTGYRFFKKIS 2452 array1.88 ISGWRLMKNIS 2453 array1.89 ASGWRLMKKES 2454array1.90 VSGWRLMKKVS 2455 array1.91 ISGWRLLKNIS 2456 array1.92ASGWRLLKKES 2457 array1.93 VSGWRLLKKVS 2458 array1.94 ISGWRLAKNIS 2459array1.95 ASGWRLAKKES 2460 array1.96 VSGWRLAKKVS 2461

Example 95 Solubility of NLpoly Variants

A single NLpoly 5A2, 12S, 11S, 125-75, 125-107 or 5P-B9 colony wasinoculated into 5 mL LB culture and incubated at 37° C. overnight withshaking. The overnight culture was diluted 1:100 into fresh LB andincubated at 37° C. for 3 hrs with shaking. Rhamnose was added to thecultures to 0.2% and incubated 25° C. overnight with shaking. 900 μl ofthese overnight cultures were mixed with 100 uL 10× FastBreak LysisBuffer (Promega Corporation) and incubated for 15 min at RT. A 75 uLaliquot (total) was removed from each culture and saved for analysis.The remaining culture from each sample was centrifuged at 14,000×rpm ina benchtop microcentrifuge at 4° C. for 15 min. A 75 uL aliquot ofsupernatant (soluble) was removed from each sample and saved foranalysis. 25 uL of 4×SDS buffer was added to the saved aliquots andincubated at 95° C. for 5 min. 5 ul of each sample was loaded onto a4-20% Tris-Glycine SDS gel and run at ˜190V for ˜50 min. The gel wasstained with SimplyBlue Safe Stain and imaged on a LAS4000. FIG. 91shows a protein gel of total lysates and the soluble fraction of thesame lysate for the NLpoly variants. With the exception of 5A2, allvariants exhibit a percentage of NLpoly in the soluble fraction.

Example 96 Solubility and Dissociation Constant of NLpoly Variants

A single NLpoly colony (listed in FIG. 92) was inoculated into 5 mL LBculture and incubated at 37° C. overnight with shaking. The overnightculture was diluted 1:100 into fresh LB and incubated at 37° C. for 3hrs with shaking. Rhamnose was added to the cultures to 0.2% andincubated 25° C. overnight with shaking. 900 μl of these overnightcultures were mixed with 100 uL 10× FastBreak Lysis Buffer (PromegaCorporation) and incubated for 15 min at RT. A 75 uL aliquot (total) wasremoved from each culture and saved for analysis. The remaining culturefrom each sample was centrifuged at 14,000×rpm in a benchtopmicrocentrifuge at 4° C. for 15 min. A 75 uL aliquot of supernatant(soluble) was removed from each sample and saved for analysis. 25 uL of4×SDS buffer was added to the saved aliquots and incubated at 95° C. for5 min. 5 ul of each sample was loaded onto a 4-20% Tris-Glycine SDS geland run at ˜190V for ˜50 min. The gel was stained with SimplyBlue SafeStain and imaged on a LAS4000.FIG. 92 shows a protein gel of total lysates and the soluble fraction ofthe same lysate for NLpoly variants as well a table containing thedissociation constants for the same variants.

Example 97 Substrate Specificity for NLpoly 5P and 11S with NLpep79

E. coli clarified lysates were prepared for NLpoly 5P or 11S asdescribed previously. The NLpoly lysates were then serially diluted insteps of 10-fold into PBS+0.1% Prionex. 25 uL NLpoly and 25 uL syntheticNLpep79 (400 nM, 4×) were mixed and incubated for 10 min at RT. 50 uLNanoGlo+100 uM Fz was added, incubated for 10 min at RT, luminescencemeasured on a GloMax Multi+ with 0.5 sec integration. FIG. 93 shows thesubstrate specificity for 5P and 11S with NLpep79 and demonstrates that11S has superior specificity for furimazine than 5P.

Example 98 Solubility of NLpoly Variants from Various Steps of Evolution

A single NLpoly WT, 5A2, 5P, 8S or 11S colony was inoculated into 5 mLLB culture and incubated at 37° C. overnight with shaking. The overnightculture was diluted 1:100 into fresh LB and incubated at 37° C. for 3hrs with shaking. Rhamnose was added to the cultures to 0.2% andincubated 25° C. overnight with shaking. 900 μl of these overnightcultures were mixed with 100 uL 10× FastBreak Lysis Buffer (PromegaCorporation) and incubated for 15 min at RT. A 75 uL aliquot (total) wasremoved from each culture and saved for analysis. The remaining culturefrom each sample was centrifuged at 14,000×rpm in a benchtopmicrocentrifuge at 4° C. for 15 min. A 75 uL aliquot of supernatant(soluble) was removed from each sample and saved for analysis. 25 uL of4×SDS buffer was added to the saved aliquots and incubated at 95° C. for5 min. 5 μl of each sample was loaded onto a 4-20% Tris-Glycine SDS geland run at ˜190V for ˜50 min. The gel was stained with SimplyBlue SafeStain and imaged on a LAS4000.

FIG. 104 shows a protein gel of total lysates and the soluble fractionof the same lysate for NLpoly variants from various steps of theevolution process. These results demonstrate that the solubility ofNLpoly was dramatically increased in the evolution process.

Example 99 Chemical Labeling of Proteins

The non-luminescent peptides (NLpeps) of the present invention can beused to chemically label proteins. An NLpep of the present invention canbe synthesized to contain a reactive group, e.g., biotin, succinimidylester, maleimide, etc., and attached (e.g., conjugated, linked, labeled,etc.) to a protein, e.g., antibody. The NLpep-labeled protein, e.g.,NLpep-antibody, can then be used in a variety of applications, e.g.,ELISA. The interaction/binding of the NLpep-labeled protein, e.g.,NLpep-antibody, to its target/binding partner would be detected byadding an NLpoly of the present invention and NanoGlo® assay reagent.The luminescence generated by the interaction of the NLpep-labeledprotein and NLpoly would correlate to the interaction of the NL-labeledprotein to its target/binding partner. This concept could allow formultiple NLpeps to be attached to a single protein molecule therebyresulting in multiple NLpep-labeled protein/NLpoly interactions leadingto signal amplification.

Example 100 Detection of Post-Translational Protein Modification UsingHaloTag-NLpep by Western Blotting

Several proteins can be posttranslationally modified by AMPylation orADP-ribosylation. In AMPylation, AMP is added to the target protein by aphosphodiester bond using ATP as the donor molecule. Similarly, inADP-ribosylation, an ADP-ribose moiety is added to target proteinsthrough a phosphodiester bond using NAD+ as the donor molecule. It hasbeen shown that the N6-position of both ATP and NAD+ can be used to taglinkers without affecting the posttranslational event. If a N6-modifiedchloroalkane-ATP or -NAD+ is used to perform the AMPylation orADP-ribosylation reaction, the target proteins would be modified tocontain the chloroalkane-ATP or -NAD+.

The N6-modified ATP/NAD has been used in combination withclick-chemistry to develop in-gel fluorescent-based detection systems.Detection of these post-translational modifications by western blottingtechniques requires antibodies, which are often not specific or notavailable. An alternative approach could be to combine the properties ofHaloTag® technology and the high luminescence of NanoLuc® luciferase(NL).

Upon post-translational modification of target proteins withchloroalkane-ATP (for AMPylation) or chloroalkane-NAD+ (forADP-ribosylation) using either cell lysate or purified proteins, samplescan be resolved by SDS-PAGE and transferred to PVDF membrane. Followingblocking, the blot can be incubated with HaloTag-NLpep. HaloTag willbind to the post-translationally-modified proteins. In the next step,the NLpoly and furimazine could be added to the blot to detect thebioluminescence. This detection method is an alternative to achemiluminescent-based approach for detection of western blots. Achemiluminescent-based approach could involve incubation HaloTag-proteinG fusions (as a primary) in the next step any secondary antibody-linkedto HRP could be used followed by ECL reaction.

Example 101 Post Translational Modification Assays

Post translational modifications (PTMs) of proteins are central to allaspects of biological regulation. PTMs amplify the diverse functions ofthe proteome by covalently adding functional groups to proteins. Thesemodifications include phosphorylation, methylation, acetylation,glycosylation, ubiquitination, nitrosylation, lipidation and influencemany aspects of normal cell biology and pathogenesis. More specifically,histone related PTMs are of great importance. Epigenetic covalentmodifications of histone proteins have a strong effect on genetranscriptional regulation and cellular activity. Examples of posttranslational modification enzymes include but not limited to,Kinases/Phosphatases, Methyltransferases (HMT)/Demethylases (HDMT),Acetyltransferases/Histone Deacetylases, Glycosyltransferases/Glucanasesand ADP-Ribosyl Transferases. Under normal physiological conditions, theregulation of PTM enzymes is tightly regulated. However, underpathological conditions, these enzymes activity can be dysregulated, andthe disruption of the intracellular networks governed by these enzymesleads to many diseases including cancer and inflammation.

The non-luminescent peptides (NLpep) and non-luminescent polypeptides(NLpoly) of the present invention can be used to determine the activityof PTM enzymes by monitoring changes in covalent group transfer (e.g.phosphoryl, acetyl) to a specific peptide substrate linked to an NLpepof the present invention. The NLpep will be linked through peptidesynthesis to small PTM enzyme specific peptide and used as a substratefor the PTM enzyme.

A) PTM Transferase Assays (HAT)

Once the PTM enzyme reaction has occurred, an aminopeptidase can be usedto degrade the non-modified peptide (NLpep; control). The modified(acetylated) peptide (NLpep-PTM enzyme substrate) would be degraded at avery slow rate or would not be degraded at all as the aminopeptidaseactivity is known to be affected by a PTM. Once the aminopeptidasereaction is complete, the NLpoly is added with the NanoGlo® assayreagent containing Furimazine. Luminescence would be generated from thesample where PTM occurred via the interaction of the NLpep and NLpoly.If no PTM occurred, the NLpep would be degraded, and no interactionbetween the NLpep and NLpoly would occur, thereby no luminescence wouldbe generated. This concept is exemplified in FIG. 197 for a generaltransferase enzyme concept and in FIG. 145 for H3K4/9acetyltransferases.

The reaction would be performed under optimal enzyme reaction conditionusing the histone peptide substrate linked to NLpep of the presentinvention and Acetyl-CoA or SAM as the acetyl or methyl group donor. Abuffer containing aminopeptidase or a mixture of aminopeptidases wouldbe added to degrade specifically all the non-modified substrates. Abuffer containing a NLpoly of the present invention and anaminopeptidase inhibitor would be added. NanoGlo® assay reagent would beadded, and luminescence detected. Luminescence generated would beproportional to the amount of non-degraded NLpep present, and thereforewould correlate with the amount of methylated or acetylated substrates,thereby indicating the amount of methyl or acetyl transferase activity.The assay can also be applied to PTM such as phosphorylation,glycosylation, ubiquitination, nitrosylation, and lipidation.

B) PTM Hydrolase Assays (HDMT)

In a similar concept to A) can be used for Histone Demethylases (HDMT).However, instead of an aminopeptidase, a PTM-specific antibody can beused to create activity interference. An NLpep of the present inventioncould be linked through peptide synthesis to small methylated peptideand used as a substrate for the hydrolase. Once a hydrolase reaction hasbeen completed, an anti-methyl antibody can be added to the reaction.This antibody will bind specifically to the methylated peptide(control). The peptide product generated by the HDMT will not bind tothe antibody. Then, an NLpoly of the present invention can be added. Ifthe antibody interferes with the interaction of NLpep and NLpoly, noluminescence will be generated. If there was hydrolysis of the PTM bythe demethylase, the NLpep and NLpoly will interact, and luminescencewill be generated. This concept is exemplified in FIG. 198 for a generalhydrolase enzyme concept and in FIG. 146 H3K4/9 demethylases.

The concept of aminopeptidase degradation of the non-modified substratecan also be used for a hydrolase assay except it would be a loss ofsignal assay instead of a gain of signal. The reaction would beperformed under optimal enzyme reaction condition using a modified(methylated or acetylated) histone peptide substrate linked to an NLpepof the present invention. A buffer containing an antibody capable ofrecognizing the methyl or acetyl group would be added. A buffercontaining an NLpoly of the present invention would be added. The NLpolywould interact with NLpep not bound to the antibody. NanoGlo® assayreagent would be added, and luminescence detected. The luminescencegenerated would be proportional to the amount of NLpep not bound to theantibody, and therefore would correlate with the amount of demethylatedor deacetylated substrate, thereby indicating the amount of demethylaseor deacetylase activity. Both hydrolase assay concepts can also beapplied to PTM hydrolases such as phosphatases, glucanases anddeubiquitinases.

In another version of these concepts, the PTM transfer or hydrolysis onthe peptide-NLpep would be alone sufficient to reduce or enhance theinteraction of NLpep with NLpoly and therefore decrease or increase theluminescence signal without the need of aminopeptidase or antibody.

The method of the present invention was used to assay a representativetransferase, the Tyrosine Kinase SRC using the following NLpep-SRCsubstrate peptide: YIYGAFKRRGGVTGWRLCERILA (SEQ ID NO: 2586). SRC enzymewas titrated in 10 μl Reaction Buffer A (40 mM Tris 7.5, 20 mM MgCl2 and0.1 mg/ml BSA) in the presence of 150 μM ATP and 2.5 μM NLpep-Srcsubstrate and incubated for 1 hour at 23° C. After incubation, 10 μl ofAmino-peptidase M (APM) reagent (40 mM Tris 7.5, 0.1 mg/ml BSA and 50 mUAPM) was added, mixed for 2 minutes on an orbital shaker, and thenincubated at 37° C. for 2 hours. To the samples, 30 μl of NLpoly Reagentwas added, and the samples were incubated at room temperature. NLpolyReagent contained the NLpoly fragment and an Aminopeptidase inhibitor.After 30 minutes, 50 μl NanoGlo® assay reagent was added and theluminescence was recorded after 3 minutes on a luminometer. It was foundthat an increase in SRC kinase enzyme activity is correlated with anincrease in luminescence over background (FIG. 199). Only backgroundactivity was found when SRC was not present indicating that thenon-phosphorylated NLpep-SRC substrate peptide was digested resulting inno light production by the NLpoly fragment, thus demonstrating use ofthe method of the present invention to monitor the activity of atransferase enzyme such as a kinase.

Example 102 Detection of Specific RNAs (Noncoding RNA or mRNA) ofInterest in Mammalian Cells, Cell Lysate or Clinical Sample

The non-luminescent peptide (NLpep) and non-luminescent polypeptide(NLpoly) of the present invention can be tethered to an RNA bindingdomain (RBD) with engineered sequence specificity. The specificity ofthe RBD can be changed with precision by changing unique amino acidsthat confers the base-specificity of the RBD. An example of one such RBDis the human pumilio domain (referred here as PUM). The RNA recognitioncode of PUM has been very well established. PUM is composed of eighttandem repeats (each repeat consists of 34 amino acids which folds intotightly packed domains composed of alpha helices). Conserved amino acidsfrom the center of each repeat make specific contacts with individualbases within the RNA recognition sequence (composed of eight bases). Thesequence specificity of the PUM can be altered precisely by changing theconserved amino acid (by site-directed mutagenesis) involved in baserecognition within the RNA recognition sequence. For detection ofspecific RNAs in the cell, PUM domains (PUM1 and PUM2) with customizedsequence specificities for the target RNA can be tethered to a NLpep andNLpoly of the present invention (e.g., as a genetic fusion protein viagenetic engineering) and can be expressed in mammalian cells. PUM1 andPUM2 are designed to recognize 8-nucleotide sequences in the target RNAwhich are proximal to each other (separated by only few base pairs,determined experimentally). Optimal interaction of PUM1 and PUM2 totheir target sequence is warranted by introducing a flexible linker(sequence and length of the linker to be determined experimentally) thatseparates the PUM and the non-luminescent peptide and non-luminescentpolypeptide. Binding of the PUM1 and PUM2 to their target sequence willbring the NLpep and NLpoly into close proximity in an orientation thatresults in a functional complex formation capable of generatingbioluminescent signal under our specific assay condition. A functionalbioluminescent complex would not be generated in the absence of the RNAtarget due to the unstable interaction of the NLpep and NLpoly pairsthat constitutes the complex.

A similar strategy can also be used for detecting RNA in clinical samplein vitro. The NLpep-PUM fusion proteins with customized RNA specificitycan be expressed and purified from suitable protein expression system(such as E. coli or mammalian protein expression system). Purifiedcomponents can be added to the biological sample along with suitablesubstrate and assay components to generate the bioluminescent signal.

Example 103 DNA Oligo-Based Detection of Specific RNA (Noncoding RNA ormRNA) in Clinical Sample or Mammalian Cell Lysate

A non-luminescent peptide (NLpep) and non-luminescent polypeptide(NLpoly) of the present invention can be attached to oligonucleotidescomplementary to the target RNA with suitable linker (amino acids ornucleotides). Functional assembly of bioluminescent complex occurs onlywhen sequence specific hybridization of DNA oligo to their target RNAbrings the NLpep and NLpoly into close proximity in an idealconformation optimal for the generation of a bioluminescent signal underthe assay conditions. The detection can also be achieved through athree-component complementation system involving two NLpeps and a thirdNLpoly. For example, two NLpep-DNA conjugates will be mixed with thetarget RNA. Functional assembly of the bioluminescent complex isachieved by subsequent addition of the third NLpoly. Thus, if adetectable signal is produced under specific assay conditions using aclinical sample or cell lysate, the presence of target RNA in such asample is inferred. Such assays are useful for detecting RNAs derivedfrom infectious agents (viral RNAs) and specific RNA biomarkers(implicated in many disease conditions such as various forms of cancers,liver diseases, and heart diseases), and could provide a new avenue fordiagnosis and prognosis of many disease conditions.

Example 104 In-Vivo Imaging

Biotechnology-derived products (Biologics), including antibodies,peptides and proteins, hold great promises as therapeutics agents.Unlike small molecule drugs, biologics are large molecules withsecondary and tertiary structures and often contain posttranslationalmodifications. Internalization, intracellular trafficking,bio-distribution, pharmacokinetics and pharmacodynamics (PK/PD),immunogenicity, etc. of biologics differ significantly from smallmolecule drugs, and there is a need for new tools to ‘track’ theseantibodies in vivo. Conventional chemical labeling with enzyme reporters(HRP, luciferase, etc.) or small fluorescent tags can significantlyalter the therapeutic value of the biologics and are not ideal for invivo imaging using biologics. Radioisotope-labeling for PET-basedimaging is also not convenient.

The NLpolys and NLpeps described herein offer a novel solution for invivo imaging of biologics. The NLpep can be genetically encoded into abiologic therapeutics without any synthetic steps. Genetic encodingallows precise control over amount of peptide per biologic molecule aswell as its position, thereby minimizing any perturbation to itstherapeutic value.

For imaging, a NLpoly along with substrate, e.g., furimazine, can beinjected into the animal. If the NLpep-biologic and NLpoly interact,luminescence would be generated. Alternatively, a transgenic animalexpressing NLpoly can be used as a model system.

Example 105 BRET Applications

This concept fundamentally measures three moieties coming together. Twoof the NLpolys and/or NLpeps form a complex, and the third moiety, whichis either fluorescent or bioluminescent, provides an energy transfercomponent. If the complex formed is bioluminescent, both bioluminescenceand energy transfer (i.e., BRET) can be measured. If the complex formedis fluorescent, the magnitude of energy transfer can be measured if thethird component is a bioluminescent molecule.

A) This example demonstrates a fluorescent dye attached to a NLpep.Alternatively, a fluorescent protein could be fused, e.g., a fusionprotein, with a NLpoly or NLpep (created from a genetic construct).

E. coli clarified lysate of NLpoly WT was prepared as describedpreviously. 40 uL NLpoly WT lysate was mixed with 10 uL of PBI-4730(NLpep1) or PBI-4877 (NLpep1-TMR) and incubated for 10 min at RT. 50 uL100 uM furimazine in 50 mM HEPES pH 7.4 was added and incubated for 30min at RT. Luminescence was measured over 400-700 nm on TECAN M1000.

FIG. 147 illustrates very efficient energy transfer from theNLPoly/NLPep complex (donor) to TMR (acceptor), and the correspondingred shift in the wavelength of light being emitted.

B) This example demonstrates using the BRET in detection, such asdetecting small molecule concentration or enzymatic activity. Becauseenergy transfer is strongly dependent on distance, the magnitude ofenergy transfer can often be related to the conformation of the system.For instance, insertion of a polypeptide that chelates calcium can beused to measure calcium concentration through modulation of energytransfer.

An enzyme that also changes the distance, either through causing aconformational change of the sensor as above or through cleavage of thesensor from the fluorescent moiety, can be measured through a system asdescribed herein. A NLpoly or NLpep bound to a fluorescent moiety givesenergy transfer when the NLpoly and NLpep interact. One example of thisis a peptide sensor that has been made wherein the NLpep is conjugatedto a fluorescent TOM dye via a DEVD linker (Caspase-3 cleavage site).When exposed to the NLpoly, energy transfer is observed. When exposed toCaspase-3, energy transfer is eliminated, but luminescence at 460 nmremains.

NLpoly 5A2 and NL-HT (NanoLuc fused to HaloTag) were purified. 20 uL of8 pM NL-HT was mixed with 20 uL of 100 nM PBI-3781 (See, e.g., U.S.patent application Ser. No. 13/682,589, herein incorporated by referencein its entirety) and incubated for 10 min at RT. 40 uL NanoGlo+100 uMfurimazine was added, and luminescence measured over 300-800 nm on TECANM1000.

20 uL of 33 ng/uL NLpoly 5A2 was mixed with 20 uL of ˜500 uM PBI-5074(TOM-NCT-NLpep). 40 uL NanoGlo+100 uM furimazine was added, andluminescence measured over 300-800 nm on TECAN M1000.

FIG. 148 illustrates energy transfer from the NLPoly/NLPep complex(donor) to TOM-dye (acceptor), and the corresponding red shift in thewavelength of light being emitted.

C) Ternary Interactions

The energy transfer with an NLpoly and NLpep can also be used to measurethree molecules interacting. One example would be a GPCR labeled withNLpoly and a GPCR interacting protein with NLpep that forms abioluminescent complex when they interact. This allows measurement ofthe binary interaction. If a small molecule GPCR ligand bearing anappropriate fluorescent moiety for energy transfer interacts with thissystem, energy transfer will occur. Therefore, the binaryprotein-protein interaction and the ternary drug-protein-proteininteraction can be measured in the same experiment. Also, thefluorescent molecule only causes a signal when interacting with aprotein pair, which removes any signal from the ligand interacting withan inactive protein (FIG. 149).

Example 106 6-Tetramethylrhodamine-PEG3-NH₂

To a solution of 6-Tetramethylrhodamine succinimidyl ester (0.25 g, 0.5mmol) in DMF (5 mL), 1-Boc-4,7,10-trioxatridecan-1,13-diamine (0.15 g,0.5 mmol) was added followed by diisopropylethylamine (0.25 mL, 1.4mmol). After stirring for 16 h, the reaction was analyzed by HPLC toconfirm complete consumption of the 6-tetramethylrhodamine succinimidylester. The reaction was concentrated to a pink film, which was dissolvedin a combination of triisopropylsilane (0.2 mL) and trifluoroacetic acid(4 mL). The pink solution was stirred for 2 h, after which analyticalHPLC confirmed complete consumption of starting material. The reactionwas concentrated to dryness to provide crude6-Tetramethylrhodamine-PEG3-NH2 as a pink film.

H-GVTGWRLCERILA-PEG-TMR (SEQ ID NO: 2578) (PBI-4877):

The fully protected peptide Boc-GVTGWRLCERILA-resin (SEQ ID NO: 2578)was synthesized by standard solid phase peptide synthesis using Fmoctechniques, then cleaved from the resin using dichloroacetic acid toliberate the fully protected peptide as a white solid. To a solution of6-Tetramethylrhodamine-PEG3-NH2 (0.05 g, 0.08 mmol) in DMF (1.5 mL),this Boc-GVTGWRLCERILA-OH (SEQ ID NO: 2578) (0.2 g, 0.07 mmol),1-hydroxyazabenzotriazole (11 mg, 0.08 mmol),1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (15 mg, 0.08 mmol) anddiisopropylethylamine (0.28 mL, 0.16 mmol) was added. After stirring for30 min, the reaction was concentrated, and the resulting crude waspartitioned between CH₂Cl₂ and water, the layers separated and theorganic layer was washed with water and brine, dried over sodium sulfateand concentrated. The resulting pink solid was dissolved in acombination of triisopropylsilane (0.2 mL) and trifluoroacetic acid (4mL). After stirring for 3 h, the reaction was concentrated, and theresulting pink film was purified with reverse phase HPLC using agradient of ACN in 0.1% aqueous TFA to provide PBI 4877 as a pinkpowder: MS (M+) calcd 2088.5, found 2089.1.

TOM-DEVDGVTGWRLCERILA-OH(SEQ ID NO: 2579) (PBI-5074):

The fully protected peptide H-DEVDGVTGWRLCERILA-resin (SEQ ID NO: 2579)was synthesized by standard solid phase peptide synthesis using Fmoctechniques. While still on the resin, a solution of 6-TOM (PBI-3739)succinimidyl ester was added and allowed to react with the freeN-terminus. The peptide was then cleaved from the resin and fullydeprotected using trifluoroacetic acid (TFA) to provide a blue solid.This solid was purified with reverse phase HPLC using a gradient of ACNin 0.1% aqueous TFA to provide PBI 5074 as a blue powder: MS (M+Z/2)calcd 1238.9, found 1238.8.

Example 107 Complementation Comparison Between a Synthetic, N-TerminalFusion and C-Terminal Fusion of NLPep78

Fusions of NLpep78-HaloTag (78-HT) and HaloTag-NLPep78 (HT-78) werequantitated, with a GST-HaloTag® fusion (GST-HT) as a control, bylabeling E. coli lysates with the HaloTag-TMR® ligand, separated bySDS-PAGE, and scanned on Typhoon. A standard curve was then createdusing known concentrations of GST-HT standard, and band intensities of78-HT and HT-78 were used to determine their concentrations.

E. coli lysates containing NLpoly11S were diluted 1:10⁷ into PBS pH7+0.1% Prionex. Serial dilutions of 78-HT, HT-78, and synthetic NLpep78were made in PBS pH 7+0.1% Prionex. 20 uL NLpoly11S and 20 uL of one ofthe NLPep were mixed and incubated at ambient temperature for 5 minutes.40 uL NanoGlo® reagent (Promega Corporation)+100 uM Fz were added, andthe samples incubates at ambient temperature for 5 min. Luminescence wasmeasured on GlomaxMulti+ using 0.5 s integration. Data was fit toone-site, specific binding using GraphPad Prism to determine Bmax andKd.

The results (FIG. 150) compare the binding of NLpoly11S to syntheticNLPep78 and NLPep78 at the N- or C-terminus of a fusion partner(HaloTag). The binding affinities were not found to changesignificantly, but Bmax was reduced when NLPep78 was at the C-terminus.

Example 108 Complementation Comparison Between a Synthetic, N-TerminalFusion and C-Terminal Fusion of NLPep79

Fusions of NLpep79-HaloTag (79-HT) and HaloTag-NLPep79 (HT-79) werequantitated, with a GST-HaloTag® fusion (GST-HT) as a control, bylabeling E. coli lysates with the HaloTag-TMR® ligand, separated bySDS-PAGE, and scanned on Typhoon. A standard curve was then createdusing known concentrations of GST-HT standard, and band intensities of79-HT and HT-79 were used to determine their concentrations.

E. coli lysates containing NLpoly11S were diluted 1:10⁷ into PBS pH7+0.1% Prionex. Serial dilutions of 79-HT, HT-79, and synthetic NLpep79were made in PBS pH 7+0.1% Prionex. 20 uL NLpoly11S and 20 uL of one ofthe NLPep were mixed and incubated at ambient temperature for 5 minutes.40 uL NanoGlo® reagent (Promega Corporation)+100 uM Fz were added, andthe samples incubates at ambient temperature for 5 min. Luminescence wasmeasured on GlomaxMulti+ using 0.5 s integration. Data was fit toone-site, specific binding using GraphPad Prism to determine Bmax andKd.

The results (FIG. 151) compare the binding of NLpoly11S to syntheticNLPep79 and NLPep79 at the N- or C-terminus of a fusion partner(HaloTag). The binding affinities were not found to changesignificantly, but Bmax was reduced when NLPep79 was at the C-terminus.

Example 109 Spectral Scan of NLpoly11S with NLPep86 Compared to PBI-4877(NLPep1-Fluorophore)

Purified NLpoly11S was diluted to 1 nM in PBS pH 7+0.01% Prionex+1 mMDTT. NLPep86 or PBI-4877 was diluted to 40 uM in PBS pH 7+0.01%Prionex+1 mM DTT. 25 uL NLpoly11S and 25 uL NLPep86 or PBI-4877 weremixed and then incubated at ambient temperature for 10 min. 50 uL buffer(PBS pH 7+0.01% Prionex+1 mM DTT)+100 uM Fz was then added. Luminescencewas measured on Tecan Infinite M1000: 300-800 nm, every 5 nm, bandwidth10 nm, gain 127, integration 0.5 s, z-position 22,000 um.

The results demonstrate (FIG. 152) that the NLPep can be conjugated tosmall molecules such as fluorescent dyes and retain interaction withNLpoly11S to produce luminescence. It also demonstrates efficient energytransfer and the ability to alter the emission spectra.

Example 110 Spectral Scan of NLpoly11S with NLPep86 Compared to PBI-5434(Fluorophore-NLPep1)

Purified NLpoly11S was diluted to 1 nM in PBS pH 7+0.01% Prionex+1 mMDTT. NLPep86 or PBI-5434 was diluted to 40 uM in PBS pH 7+0.01%Prionex+1 mM DTT. 25 uL NLpoly11S and 25 uL NLPep86 or PBI-5434 weremixed and then incubated at ambient temperature for 10 min. 50 uL buffer(PBS pH 7+0.01% Prionex+1 mM DTT)+100 uM Fz was then added. Luminescencewas measured on Tecan Infinite M1000: 300-800 nm, every 5 nm, bandwidth10 nm, gain 127, integration 0.5 s, z-position 22,000 um.

The results demonstrate (FIG. 153) that the NLPep can be conjugated tosmall molecules such as fluorescent dyes and retain interaction with 115to produce luminescence. This, along with the results with PBI-4877 inExample 109, also suggests that the terminus and/or the linker lengthused for the conjugation can significantly affect the energy transfer.

Example 111 Spectral Scan of NLpoly11S with NLPep86 Compared to PBI-5436(Fluorophore-NLPep1)

Purified NLpoly11S was diluted to 1 nM in PBS pH 7+0.01% Prionex+1 mMDTT. NLPep86 or PBI-5436 was diluted to 40 uM in PBS pH 7+0.01%Prionex+1 mM DTT. 25 uL NLpoly11S and 25 uL NLPep86 or PBI-5436 weremixed and then incubated at ambient temperature for 10 min. 50 uL buffer(PBS pH 7+0.01% Prionex+1 mM DTT)+100 uM Fz was then added. Luminescencewas measured on Tecan Infinite M1000: 300-800 nm, every 5 nm, bandwidth10 nm, gain 127, integration 0.5 s, z-position 22,000 um.

The results demonstrate (FIG. 154) that the NLPep can be conjugated tosmall molecules such as fluorescent dyes and retain interaction with 11Sto produce luminescence. It also demonstrates efficient energy transferand the ability to alter the emission spectra.

Example 112 Comparison of Km Values for 11S with Various NLPeps inAffinity Buffer

Purified NLpoly11S was diluted to 40 pM in PBS pH 7+0.01% Prionex+1 mMDTT+0.005% Tergitol (affinity buffer) or NanoGlo assay reagent (PromegaCorporation). NLPeps (NLpep86, 78, 99, 101, 104, 128 and 114) werediluted to 400 uM (NLPep to 1 mM) in affinity buffer or NanoGlo assayreagent. 300 uL NLpoly11S and 300 uL of an NLPep were mixed andincubated at ambient temperature for 30 min. 50 μl was then added to awell of white 96-well plates. 50 μl affinity buffer+2x Fz (12.5 uMdiluted 2-fold 7 times) or 50 ul NanoGlo+2x Fz (100 uM diluted 2-fold 7times) was added to each well, and luminescence measured on a GlomaxMulti+ using 0.5s integration. Km was determined using GraphPad Prism,Michaelis-Menten.

The results demonstrate substrate binding in affinity buffer (FIG. 155)or NanoGlo assay buffer (FIG. 156) to the complex between NLpoly11S andvarious NLPeps. The determined Km values do not fluctuate significantlywith the indicated NLPeps.

Example 113 NLPep1 Binding Affinity to NLpoly11S at VariousConcentrations of Furimazine

Purified NLpoly156 and NLpoly11S to 40 pM in affinity buffer (PBS pH7+0.01% prionex+1 mM DTT+0.005% tergitol). Synthetic NLPep1 (WT) wasdiluted to 560 uM for NLpoly156 or 80 uM for NLpoly11S in affinitybuffer and then serially diluted 3-fold to make 8 concentrations. 350 uLNLPep1 and 350 uL NLPoly156 or 11S were mixed and then incubated atambient temperature for 30 min. 50 uL was then aliquoted into a well ofwhite 96-well assay plate. Fz was added to affinity buffer to 40, 20,10, 5, 2.5 and 1.25 uM, 50 uL Fz/affinity buffer added to each well andincubated at ambient temperature for 2 min. Luminescence was measured ona Glomax Multi+ with 0.5 s integration. GraphPad Prism and 1-sitespecific binding was used to calculate Kd at each concentration of Fz.

The results (FIG. 157) indicate the change in affinity (NLPoly/NLPep)with increasing concentrations of Fz.

Example 114 Furimazine Km Values for NLpoly156/NLPep1 andNLpoly11S/NLPep1 at Various Concentrations of NLPep1

Purified NLpoly156 and NLpoly11S were diluted to 40 pM in affinitybuffer (PBS pH 7+0.01% prionex+1 mM DTT+0.005% tergitol). SyntheticNLPep1 (WT) was diluted to 560 uM for NLpoly156 or 80 uM for NLpoly11Sin affinity buffer and then serially diluted 3-fold to make 8concentrations. 50 uL was then aliquoted into a well of white 96-wellassay plate. Fz was added to affinity buffer to 40, 20, 10, 5, 2.5 and1.25 uM, 50 uL Fz/affinity buffer added to each well and incubated atambient temperature for 2 min. Luminescence was measured on a GlomaxMulti+ with 0.5 s integration. GraphPad Prism and 1-site specificbinding was used to calculate Kd at each concentration of NLPep1.

The results (FIG. 158) indicate the change in affinity (NLPoly/NLPep)with increasing concentrations of NLPep 1.

Example 115 Comparison of Maximal Activity for NLPoly156/NLPep1,NLPoly11S/NLPep1, and NanoLuc® Luciferase

Purified NLPoly156, NLPoly11S, or NanoLuc® luciferase (Nluc) werediluted to 40 pM in affinity buffer (PBS pH 7+0.01% prionex+1 mMDTT+0.005% tergitol). Synthetic NLPep1 (WT) was diluted to 560 uM forNLPoly156 or 80 uM for NLPoly11S in affinity buffer and then seriallydiluted 3-fold to make 8 concentrations. 350 uL NLPep1 (or affinitybuffer) and 350 uL NLPoly (or Nluc) were mixed and then incubated atambient temperature for 30 min. 50 uL was then aliquoted into a well ofwhite 96-well assay plate. Fz was added to affinity buffer to 40, 20,10, 5, 2.5 and 1.25 uM, 50 uL Fz/affinity buffer added to each well andincubated at ambient temperature for 2 min. Luminescence was measured ona Glomax Multi+ with 0.5 s integration. GraphPad Prism andMichaelis-Menton equation was used to calculate Vmax at eachconcentration of NLPep (input calculated Vmax values at eachconcentration of NLPep1 into 1-site specific binding to calculate Bmax).GraphPad Prism and 1-site specific binding was used to calculate Bmax ateach concentration of Fz (input calculated Bmax values at eachconcentration of Fz into Michaelis-Menton equation to calculate Vmax).

The results (FIG. 159) demonstrate the maximal activity of NLPoly156 orNLPoly11S upon activation by NLPep1 to the maximal activity of NanoLucluciferase.

Example 116 Luminescent Values Resulting from Titrating NLpoly11S withVarious NLPeps

Purified NLPoly11S was diluted to 40 pM in PBS pH 7+0.01% Prionex+1 mMDTT+0.005% Tergitol (affinity buffer). Synthetic NLPeps (NLPep86, 78,79, 99, 101, 104, 114, 128 or wt) were diluted in affinity buffer asfollows: NLPep86=60 nM, NLPep78=280 nM, NLPep79=800 nM, NLPep99=4 uM,NLPep101=34 uM, NLPep104=20 uM, NLPep128=4 uM, NLPep114=4.48 mM andNLPepWT=20 uM. 25 uL NLPoly11S and 25 uL an NLPep were mixed and thenincubated at ambient temperature for 30 min. 50 μl affinity buffer+20 uMFz was then added to each mixture, and luminescence measured on aGlomaxMulti+ using 0.5 s integration. Bmax and Kd values were determinedusing GraphPad Prism and 1 site specific binding.

The results (FIG. 160) demonstrate ˜100,000-fold range of affinitiesusing NLPoly11S and various NLPeps. Minimal loss in Bmax was observedbetween the high affinity and low affinity NLPeps.

Example 117 Western Blot of NLPoly156, NLPoly11S, and NanoLuc®Luciferase after Transfection into HEK293T Cells

On day 1, a transfection mixture of 2 ng NLPoly156, NLPoly 11 S orNanoLuc® luciferase (Nluc) DNA, 1 ug pGEM3Zf(+) carrier DNA, 4 μl FugeneHD (Promega Corporation) and Phenol red-free OptiMEM to 100 μl was madeand incubated at RT for 10 minutes. The transfection mixture was thentransferred to one well of 6 well plate, and 2 ml of HEK293T cells at400,000 cells/ml (800,000 cells total) was added. The cells wereincubated overnight at 37° C.

On day 2, the cells were washed with phenol red-free DMEM, 500 uL phenolred-free DMEM added to each well, and the cells frozen at −70° C. for atleast 30 min. The cells were then thawed, 500 uL transferred tomicrocentrifuge tube, and 20 μl mixed with 80 uL of 1.25×SDS loadingbuffer and incubated at 95° C. for 5 min. 10 ul was loaded onto 10%Bis-Tris NuPAGE gel with MES running buffer. Protein was transferred toPVDF using iBlot, and the membrane washed in methanol. The membrane wasthen blocked in TBST+5% BSA for 1 hr at ambient temperature, washed 3times in TBST and then incubated with 10 mL TBST+2 uL rabbit anti-Nlucpolyclonal antibody+2 uL rabbit anti-β-actin polyclonal antibody (Abcam#ab8227) at 4° C. overnight.

On day 3, the membrane was washed 3 times in TBST, incubated with 10 mLTBST+2 uL anti-rabbit HRP conjugated antibody for 1 hr at ambienttemperature, washed again 3 times with TBST and incubated with 12 mL ECLWestern Blotting Substrate for 1 min. Chemiluminescence was imaged withLAS 4000 Image Quant.

The results (FIG. 161) show the expression level of NLPoly compared tofull-length NanoLuc® luciferase. NLPoly156 does not express as well asNanoLuc® luciferase (Nluc), whereas NLPoly11S expresses similarly toNluc.

Example 118 Determination of the Influence of NLPoly11S/NLPep114Affinity on the Interaction Between a β-lactamase (SME) and β-LactamaseInhibitory Protein (BLIP) and Comparison Between Affinity ValuesMeasured Through 11S/114 and β-Lactamase Activity Protein Purification

pF1K-signal-6H-SME, pF1K-signal-6H-SME-11S, pF1K-signal-6H-BLIPY50A, andpF1K-signal-6H-BLIPy50A-114 (Promega Flexi vectors for T7 promoter-basedexpression of recombinant protein in E. coli; the signal refers to thenative signal peptide for either SME or BLIP) were induced with rhamnoseto express in the periplasm of KRX cells at 25° C. for 18-20 hrs. Cellswere pelleted and resuspended in B-Per lysis reagent (Pierce; 1/50thculture volume) and incubated at ambient temperature for 15 min. Lysatewas then diluted by addition of 1.5× volume 20 mM Tris pH 8+500 mM NaCland centrifuged at 12,000×g for 10 min. The supernatant was transferredto a clean tube, 1 mL RQ1 DNase (Promega Corporation) added andcentrifuged again at 12,000×g for 10 min. Supernatant was purified overHisTALON column Clontech) with 25 mM Tris pH 8 and 500 mM NaCl loadingbuffer and eluted with 25 mM Tris pH 8, 500 mM NaCl and 50 mM imidazole.Eluted protein was dialyzed into 25 mM Tris pH 7.5 and 25 mM NaCl andpurified over HiTrap Q FF column (GE Healthcare) with 25 mM Tris pH 7.5and 25 mM NaCl loading buffer and eluted with 25 mM Tris pH 7.5 and 125mM NaCl. Ionic strength was adjusted to final concentration of 150 mMNaCl, and concentrated using a VivaSpin concentrator.

Assay

BLIPY50A and BLIPY50A-114 were diluted to 312.5 nM in affinity buffer(PBS pH 7 0.01% prionex 0.005% tergitol 1 mM DTT), and then seriallydilute 1.5-fold. SME and SME-11S were diluted to 0.2 nM in affinitybuffer. 11.11 uL SME and 88.89 uL BLIP were mixed and then incubated atambient temperature for 2 hrs. 90 uL of the mixture was transferred to aclear 96-well plate with 10 uL of 100 uM Nitrocefin (Calbiochem inaffinity buffer). 90 uL of SME-11S/BLIPY50A-114 was transferred to awhite 96-well plate with 10 uL of 100 uM Fz (in affinity buffer).Absorbance (nitrocefin) was measured at 486 nm every 15 sec over 30 min,and luminescence (Fz) was measure every 2 min over 30 min.

For nitrocefin, initial velocities were fit using Excel. Initialvelocities vs. BLIP concentration were plotted. Fit Ki usingE_free=[E]−([E_0]+[I_0]+K_app−√((([E_0]+[I_0]+K_app)̂2−(4[E_0][I_0])))/2and K_app=K_i(1+([S])/K_M) For Fz, Kd usingRLU=(Bmax×[BLIP−114])/([BLIP−114]+K_D) was fit.

The results (FIG. 162) compares the affinity of a protein interaction(the β-lactamase SME and its inhibitor BLIPY50A) as unfused proteins tothe affinity when NLPoly and NLPep are fused to them and demonstratesthe affinity between NLPoly11S and NLPep114 does not result in anincreased apparent affinity for the SME/BLIPY50A interaction. This alsodemonstrates the use of NLPoly11S and NLPep114 to measure an equilibriumbinding constant for a protein interaction, and the affinity measuredthrough NLPoly11S and NLPep114 is consistent with the affinity measuredby activity of the target protein (SME).

Example 119 Comparison of Luminescence Generated by Cells ExpressingDifferent Combinations of FRB-NLPoly11S with FKBP-NLPep101 and 111-136

HEK293T cells (20,000) were reverse-transfected into wells of opaque96-well assay plates with a total of 1 ng pF4A Ag FRB-NLpoly11S and pF4AAg FKBP-NLpep101 or 111-136 plasmid DNA using FuGENE HD at aDNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring totalDNA in each transfection to 1 μg. Twenty-four hours-post transfection,cells were washed with PBS and then incubated in phenol red-freeOptiMEMI with or without 50 nM rapamycin for 1.5 h. 10 μM furimazinesubstrate (final concentration) with or without 50 nM rapamycin inOptiMEMI was added directly to each well and incubated at roomtemperature for 5 min. Luminescence was then read on a GloMax Multi with0.5 s integration time.

FIG. 163 demonstrates that, of tested combinations, NLpoly11S withNLpep114 shows the greatest rapamycin induction and one of the strongestrapamycin-specific luminescent signals.

Example 120 Comparison of Luminescence Generated by Cells ExpressingDifferent Combinations of FRB-NLpoly11S with FKBP-NLpep114 and 137-143

HEK293T cells (20,000) were reverse-transfected into wells of opaque96-well assay plates with a total of 1 ng pF4A Ag FRB-NLpoly11S and pF4AAg FKBP-NLpep114 or 137-143 plasmid DNA using FuGENE HD at aDNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring totalDNA in each transfection to 1 μg. Twenty-four hours post transfection,cells were washed with PBS and then incubated in phenol red-freeOptiMEMI with or without 50 nM rapamycin for 1.5 h. 10 μM furimazinesubstrate (final concentration) with or without 50 nM rapamycin inOptiMEMI was added directly to each well and incubated at roomtemperature for 5 min. Luminescence was then read on a GloMax Multi with0.5 s integration time.

FIG. 164 demonstrates that, of tested combinations, NLpoly11S withNLpep114 shows the greatest rapamycin induction and one of the strongestrapamycin-specific luminescent signals.

Example 121 Rapamycin Dose Response Curves of Cells ExpressingFRB-NLpoly11S and FKBP-NLpep78/79/99/101/104/114/128

HEK293T cells (20,000) were reverse-transfected into wells of opaque96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S andpF4A Ag FKBP-NLpep78/79/99/101/104/128 plasmid DNA using FuGENE HD at aDNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bring totalDNA in each transfection to 1 μg. Twenty-four hours post transfection,cells were washed with PBS and then incubated in phenol red-freeOptiMEMI with 0 to 300 nM rapamycin for 2 h. 10 μM furimazine substrate(final concentration) with 0 to 300 nM rapamycin in OptiMEMI was addeddirectly to each well and incubated at room temperature for 5 min.Luminescence was then read on a GloMax Multi with 0.5 s integrationtime. Graphpad Prism was used to fit data to sigmoidal curve andcalculate EC50 values.

FIG. 165 shows a sigmoidal dose response to rapamycin for NLpoly11S withNLpep78/79/99/101/104/114/128. Of the combinations plotted, NLpoly11Swith NLpep114 shows the greatest dynamic range.

Example 122 Response of Cells Expressing FRB-NLpoly11S andFKBP-78/79/99/101/104/114/128 to the Rapamycin Competitive InhibitorFK506

HEK293T cells (20,000) were reverse-transfected into wells of opaque96-well assay plates with a total of 0.1 ng pF4A Ag FRB-NLpoly11S andpF4A Ag FKBP-NLpep78/79/99/101/104/114/128 plasmid DNA using FuGENE HDat a DNA-to-FuGENE ratio of 1 to 8. pGEM-3Zf(+) DNA was added to bringtotal DNA in each transfection to 1 μg. Twenty-four hours posttransfection, cells were washed with PBS and then phenol red-freeOptiMEMI with 10 nM rapamycin was added for 2 h. FK506 inhibitor inOptiMEM was added to cells at final concentrations of 0 to 50 μM andincubated for 3 h. Furimazine in OptiMEM was added to cells for a finalconcentration of 10 μM on cells. Luminescence was immediately read on aGloMax Multi with 0.5 s integration time. Graphpad Prism was used toplot data, fit to a sigmoidal curve, and calculate IC50 values.

FIG. 166 demonstrates dose-dependent decreases in rapamycin-inducedsignal of FRB-NLpoly11S and FKBP-78/79/99/101/104/114/128 with therapamycin competitive inhibitor, FK506.

Example 123 Comparison of Luminescence Generated by Cells Transfectedwith Different Ratios of FRB-NLpoly11S and FKBP-NLpep114

HEK293T cells (20,000) were reverse-transfected into wells of opaque96-well assay plates with 1 ng pF4A Ag FRB-NLpoly11S and 0.01, 0.1, 1,10, or 100 ng pF4A Ag FKBP-NLpep114 plasmid DNA using FuGENE HD at aDNA-to-FuGENE ratio of 1 to 8. HEK293T cells (20,000) were also reversetransfected with 1 ng pF4A Ag FKBP-NLpep114 and 0.01, 0.1, 1, 10, or 100ng pF4A Ag FRB-NLpoly11S. In both situations, pGEM-3Zf(+) DNA was addedto bring total DNA in each transfection to 1 μg. Twenty-four hours posttransfection, cells were washed with PBS and then incubated in phenolred-free OptiMEMI with or without 50 nM rapamycin for 1.5 h. 10 μMfurimazine substrate (final concentration) with or without 50 nMrapamycin in OptiMEMI was added directly to each well and incubated atroom temperature for 5 min. Luminescence was then read on a GloMax Multiwith 0.5 s integration time.

FIG. 167 demonstrates that a DNA ratio of 1:1 generated the greatestrapamycin induction, although a significant induction was observed atall DNA ratios tested.

Example 124 Comparison of Luminescence Generated by Cells ExpressingNLpoly11S/NLpep114 Fusions of FRB/FKBP in Different Orientations andwith Different Linker Lengths

HEK293T cells (20,000) were transfected into wells of 96-well plateswith vectors expressing combinations of N- and C-terminal fusions ofpF4Ag NLpoly11S and pF4Ag NLpep114 with FRB or FKBP. In theseconstructs, NLpoly11S/NLpep114 were separated from their fusion partnerswith either a 4, 10, or 15 serine/glycine linker. 0.1 ng NLpoly11S andNLpep114 DNA was transfected per well at a DNA-to-FugeneHD ratio of 1 to8. Twenty-four hours post transfection, cells were washed with PBS andthen incubated in phenol red-free OptiMEMI with or without 50 nMrapamycin in OptiMEMI for 2 h. 10 μM Furimazine substrate was thenadded, and following a 5 min incubation at room temperature, the platewas read using a GloMax Multi with 0.5 s integration time.

FIG. 168 illustrates a rapamycin-specific increase in RLU regardless offusion orientation or linker length.

Example 125 Comparison of Rapamycin Dose Response Curve and Time CourseGenerated by FRB-NLpoly11S/FKBP-NLpep114 and Split FireflyComplementation Systems

HEK293T cells (800,000) were transfected into wells of 6-well plateswith a total of 20 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep114 or750 ng pF4A Ag N-Fluc(1-398)-FRB and FKBP-C-Fluc(394-544) using FuGENEHD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was added tobring total DNA in each transfection to 1 ug. Twenty-four hours posttransfection, 20,000 cells were re-plated into wells of opaque 96-wellassay plates and incubated an additional 24 h.

For dose response experiments (FIG. 169A), NLpoly11S/NLpep114-expressingcells were treated with 0-1 μM rapamycin in phenol red-free OptiMEMI for3 h and then incubated with 1004 furimazine for 5 min before recordingluminescence on GloMax Multi. Cells expressingN-Fluc(1-398)/C-Fluc(394-544) were incubated with 0-1 μM rapamycin inphenol red-free for 2 h, followed by an additional 1 h incubation in thepresence of 4 mM D-Luciferin, prior to recording luminescence on GloMaxMulti.

For time course experiments (FIG. 169B), NLpoly11S/NLpep114-expressingcells were treated with 0 or 50 nM rapamycin in phenol red-free OptiMEMIwas added via GloMax Multi injector, and luminescence was immediatelymeasured. Cells expressing N-Flu(1-398)/C-Flu(394-544) were treated with4 mM D-luciferin in phenol red-free OptiMEMI for 1 h followed byaddition of 0 or 50 nM rapamycin via injector and measurement ofluminescence by GloMax Multi. Curves were fit using GraphPad Prism 6software. FIG. 169A-B demonstrate that both NLpoly11S/NLpep114 and splitfirefly complementation systems respond in a rapamycin-dependent manner,generating sigmoidal dose response curves and similar EC50 values. TheNLpoly11S/NLpep114 system displays faster association kinetics and ahigher maximum signal.

Example 126 Comparison of FK506 Dose Response Curve and Time CourseGenerated by FRB-NLpoly11S/FKBP-NLpep114 and Split FireflyComplementation Systems

HEK293T cells (800,000) were transfected into wells of 6-well plateswith a total of 20 ng pF4A Ag FRB-NLpoly11S and pF4A Ag FKBP-NLpep114 or750 ng pF4A Ag N-Fluc(1-398)-FRB and FKBP-C-Fluc(394-544) using FuGENEHD at a DNA-to-FuGENE ratio of 1 to 4. pGEM-3Zf(+) DNA was added tobring total DNA in each transfection to 1 ug. Twenty-four hours posttransfection, 20,000 cells were re-plated into wells of opaque 96-wellassay plates and incubated an additional 24 h. Cells were then treatedwith 0 or 20 nM rapamycin in phenol red-free OptiMEMI for 3 h.

For FK506 dose response experiments (FIG. 170A), cells were incubatedwith 0 to 100 μM FK506 inhibitor in phenol red-free OptiMEMI for 5 h,treated with 10 μM furimazine, and then read with GloMax Multi inluminescence mode with 0.5 s integration time. For time courseexperiments (FIG. 170B), cells were treated with 10 μM FK506 in phenolred-free OptiMEMI containing 10 μM furimazine and luminescence wasimmediately read with GloMax Multi.

FIG. 170A-B demonstrates that the NLpoly11S/NLpep114 and split fireflycomplementation systems show a dose-dependent decrease in light outputfollowing treatment with the FK506 inhibitor. The loss of signal in theNLpoly11S/NLpep114 system begins at an earlier time point, is morerapid, and is more complete than the split firefly system.

Example 127 Western Blot Showing Expression Levels of FKBP-NLpep114 andFKBP-Fluc(394-544)

HEK293T cells (200,000) were transfected with 0 to 30 ng of pF4AgNLpep114-FKBP or pF4Ag FKBP-Fluc(394-544) DNA using FugeneHD at a DNA toFugene ratio of 1 to 8. Forty-eight hours post-transfection, cells wereharvested with 1×SDS gel loading buffer. Samples were separated on a4-10% Tris-HCl SDS-PAGE gel and transferred to PVDF membrane. Themembrane was blocked in 5% BSA in TBST for 1 h and then incubated withanti-FKBP (Abcam #ab2918) overnight. Secondary antibody incubation withhorse radish peroxidase-conjugated donkey anti-rabbit IgG was performedfor 1 h and then the blot was developed using ECL Western BlottingSubstrate (Promega Corporation) and the Image Quant LAS 4000 system.

FIG. 171 demonstrates similar expression levels of FKBP-NLpep114 andFKBP-Fluc(394-544) at equal levels of transfected DNA.

Example 128 Dose- and Time-Specific Inhibition of NLpoly11S-BRD4 andHistone H3.3-NLpep114 Interaction by IBET-151

HEK293T cells (20,000) were transfected into wells of a 96-well whiteassay plate with 10 ng of pF4Ag Histone H3.3-NLpep114 andNLpoly11S-NLpoly11S using Fugene HD at a DNA to Fugene ratio of 1 to 8.

For dose response experiment (FIG. 172A), cells were treated with 0 to10 μM IBET-151 in phenol red-free OptiMEMI for 4 h at 37° C. and thentreated with 10 μM furimazine for 5 min before reading luminescence withGloMax Multi.

For time course experiment (FIG. 172B), cells were pre-incubated with 10μM furimazine for 5 min, treated with 0-500 nM IBET-151 and immediatelyplaced in a GloMax Multi for luminescent measurements every 5 min.

FIG. 172A-B demonstrates a dose-dependent decline in luminescence upontreatment with the BRD4 inhibitor IBET-151 that occurs within 3 hours oftreatment, consistent with literature reports.

Example 129 RAS/CRAF, BRAF/BRAF, and CRAF/BRAF Dimerization in Responseto GDC0879

HEK293T cells (20,000) were co-transfected into wells of 96-well assayplates with combinations of pF4Ag NLpoly11S-BRAF, NLpoly11S-CRAF,NLpep114-KRAS, or NLpep114-BRAF using a total of 0.1 ng DNA per well andFugene HD at a ratio of 1 to 4. Twenty-four hours post-transfection,cells were treated with 0 to 10 μM of the BRAF inhibitor GDC0879 inphenol red-free OptiMEMI for 4 h. Furimazine substrate in phenolred-free OptiMEMI was added to 10 μM, and luminescence was readimmediately with GloMax Multi set to 0.5 s integration time.

FIG. 173 demonstrates a dose dependent increase of RAS/CRAF, BRAF/BRAFand CRAF/BRAF dimerization in response to BRAF inhibitor GDC0879.

Example 130

Twelve synthetic peptides (FIG. 180) were examined for their ability tostructurally complement three different versions of NLpoly11S (i.e. 11S,11S-amino acid 157, 11S-amino acids 156 and 157). Stocks of NLpoly weremade to 35 nM in NanoGlo reagent and stocks of NLpep were made to 12.5nM in PBS pH 7.2. Equal volumes were mixed and samples measured forluminescence on a Tecan Infinite F500 reader (100 msec integration time;10 min time point) (FIG. 200).

Example 131 Spontaneously Interacting Peptide NLpep86

Purified NLPoly11S was diluted to 40 pM in PBS pH 7+0.01% Prionex+1 mMDTT+0.005% Tergitol (affinity buffer). Synthetic NLPeps (NLPep86, WT,114) were diluted in affinity buffer as follows: NLPep86=60 nM,NLPep114=4.48 mM and NLPepWT=20 uM. 25 uL NLPoly11S and 25 uL an NLPepwere mixed and then incubated at ambient temperature for 30 min. 50 μlaffinity buffer+20 uM Fz was then added to each mixture, andluminescence measured on a GlomaxMulti+ using 0.5 s integration. Bmaxand Kd values were determined using GraphPad Prism and 1 site specificbinding.

FIG. 174 demonstrates 100,000-fold range of affinities using NLPoly11Sand various NLPeps. Pep 86 is an example of a spontaneously interactingpeptide (with LSP 11S), and Pep 114 is shown for reference as a lowaffinity interacting peptide.

Example 132 Titration of High Affinity Peptide In Vitro

Purified NLpoly11S (HaloTag purification/E. coli expression; pFN18K) andsynthetic peptide NLpep86 (obtained from Peptide 2.0) were titrated at alinear dynamic range using 33 nM NLpoly11S in Nano-Glo® assay buffer to3.3 fM-100 nM high affinity NLpep86. For a 30 kDa protein, thiscorresponds to LOD of 10 fg.

FIG. 176 demonstrates the broad linear range and ability to detectfemptamolar concentrations of the high affinity peptide tag (NLpep86).This rivals most sensitive Western Blot (WB)+Enhanced Chemiluminescence(ECL) kits

Example 133 Western Blot-Like Utility of NLpoly and NLpep

A titration of HaloTag (HT7)-NLpep 80 (80) or NLpep80-HaloTag (HT7) wererun on an SDS page gel. The HaloTag® protein was imaged with HaloTag-TMRligand (Promega Corporation) on a Typhoon scanner. The samples weretransferred to a membrane and PBS pH 7+0.1% Prionex+NLpoly11S (E. colilysate diluted 1:1,000) was used to blot the membrane. NanoGlo/Fz wasthen added to the membrane and it was imaged on a ImageQuant.

FIG. 177 demonstrates the sensitivity of detecting proteins tagged witha high affinity NLPep using NLpoly11S. FIG. 177 also compares thedetection using NLPep/NLPoly to the detection using fluorescentlylabeled HaloTag.

Example 134 Stability of an NLpoly11S Reagent

100 nM NLpoly11S was incubated in NanoGlo assay buffer (PromegaCorporation)+100 uM furimazine and assayed with equal amounts of dilutedNLpep86. As a control, NanoGlo assay buffer+100 uM furimazine was usedto assay an equal volume of diluted NanoLuc® luciferase (PromegaCorporation).

The results (FIG. 178) demonstrate that an NLpoly11S reagent (containingFz) has similar stability compared to the commercial NanoGlo® assayreagent (also containing Fz).

Example 135 Titration of DNA for High Affinity NLpep78-HT7 Fusion

HEK293 cells (200,000/ml) were reverse transfected with 10-folddilutions of DNA (starting with 100 ng) from a high affinity peptide,NLpep78, fused to HaloTag® protein (HT7). 100 μl of each transfectionwas plated in triplicate into wells of a 96-well plate. Twenty-fourhours post-transfection, 100 μl NanoGlo® assay buffer containing 100 nMNLpoly11S and 100 μl furimazine was added and mixed. Luminescence wasmeasured 10 minutes after reagent addition on a GloMax luminometer.

The results (FIG. 179) demonstrate the broad linear range similar toExample 131/FIG. 27. This is essentially a similar experiment to whatwas done in Example 131 except that this examples uses recombinantlyexpressed peptide (fused to HaloTag) in a mammalian cell.

Example 136 Preliminary Results (Array Peptides)

In FIG. 183A, 50 nM NLpoly11S was mixed with 7.5 μM NLpep114 and 37.5 μMdark peptide (DP) candidate (Q-162, A-162, K-162 or E-162). NanoGlo®assay reagent (Promega Corporation) was added and incubated for 5minutes. Luminescence was detected. In FIG. 183B, 50 nM NLpoly11S inassay buffer (PBS pH7+0.01% Prionex+1 mM DTT+0.005% Tergitol) was mixedwith 7.5 μM NLpep114 (also in assay buffer) and variable amounts of darkpeptide (DP) candidates Q-162 or K-162 (also in assay buffer). NanoGlo®assay reagent (Promega Corporation) was added and incubated for 5minutes. Luminescence was detected on a Tecan Infinite F500 reader; 100ms integration time; 5 min time point used.

Panel A indicates that each of the peptide candidates (at 7.5 uM) caninhibit the binding between NLpoly11S and NLpep114, as indicated by lessbioluminescence. Note these “dark” peptides do generate someluminescence, thus the increased signal compared to no peptides at all.

Panel B indicates that with the Lys-162 and Gln-162 peptides theinhibition is dose-dependent.

Example 137 High Purity (>95%) Dark Peptides

In FIG. 184A, 5 nM NLpoly11S was mixed with 500 nM NLpep114 and variableamounts of a dark peptide (DP) candidate Q-162 or A-162 (n=3). NanoGlo®assay reagent (Promega Corporation) was added and incubated for 5minutes. Luminescence was detected.

In FIG. 184B, 5 nM NLpoly11S in assay buffer was mixed with variableamounts of dark peptide (DP) candidates Q-162 or A-162 in assay buffer(no NLpep114)(n=3). NanoGlo® assay reagent (Promega Corporation) wasadded and incubated for 5 minutes. Luminescence was detected.

The results (FIGS. 184A and B) substantiate the results from Example135, but there is greater confidence here because the peptides are morepure. These results also suggest that of the dark peptides variantstested the Ala peptide is the most potent as an inhibitor.

Example 138 Inhibition of Circularly Permuted NanoLuc® Luciferase byDark Peptides

To determine whether “high affinity/low activity” NLpeps (a.k.a. DarkPeptides) can compete with the intramolecular interaction (i.e., proteinfolding) between NanoLuc® luciferase (Nluc) residues 1-156 and 157-169in the context of circularly permuted Nluc (CP Nluc).

CP NLuc: NLuc 157-169-33aa-linker-Nluc 1-156 (SEQ ID NO: 2366)Dark peptides: VTGWRLCERIL (wt) (SEQ ID NO: 2388) 1. Gln-162 VSGWQLFKKIS(SEQ ID NO: 2390) 2. Ala-162 VSGWALFKKISRecombinant CP Nluc was prepared as a soluble fraction of an E. coli5×-concentrated lysate (T7-promoter; overnight expression). A10,000-fold dilution of the CP Nluc in Assay Buffer (PBS pH 7/0.01%Prionex/1 mM DTT/0.005% Tergitol) was used. Synthetically-derived darkpeptides were prepared across a range of concentrations, also in theAssay Buffer. Reactions were set up using 30 μL of CP NLuc and 604 ofDark peptide and assayed by adding 904 NanoGlo® assay reagent (PromegaCorporation). Luminescence was measured (5 min) on a Tecan Infinite F500reader (100 ms integration). Three replicates were used for Dark peptidesamples. Two replicates were used for buffer controls (acetic acid frompeptide stocks).

FIG. 185 demonstrates a dose-response of the dark peptides with CP Nluc.FIG. 186 demonstrates a time course of dark peptide (56 μM peptide) withCP Nluc.

The results indicate that both dark peptides, particularly the Ala162version, are able to significantly inhibit generation of luminescence byCP Nluc (Ala162>2 logs; Gln162>1 log). This indicates that a CP Nlucapproach has utility for inverse complementation.

Example 139 Dark Peptides in Cells

In this example, the following constructs were used:

-   -   Four dark peptide vectors: pF4Ag+FKBP-dark peptide Ala-162,        Leu-162, Gln-162 and Val-162    -   Two non-dark peptide vectors: pFc5K2 FKBP-NLpep114 (low affinity        peptide) and pFc5K2 FKBP-NLpep80 (high affinity peptide)    -   One NLpoly vector: pFc5K2 FRB-NLpoly11S        All constructs harbored a CMV promoter for mammalian cell        expression. All fusions constructs contained a 10aa Gly-Ser        flexible linker.

Serial dilutions of the dark peptide constructs, Ala-162 (A), Leu-162(L), Gln-162 (Q) and Val-162 (V), were made in OptiMem and additionallycontained carrier DNA (pGEM-3Z).

For transfection containing NLpoly11S only, 20 μl of diluted darkpeptide was mixed with 20 ul NLpoly11S, 60 μl OptiMem and 8 uL Fugene.For transfections containing NLpoly11S and NLpep114 or NLpep80, 20 μl ofdiluted dark peptide was mixed with 20 ul NLpoly11S (10 ng/ul), 20 ulNLpep114 or NLpep80 (10 ng/ul), 40 μl OptiMem and 8 μl Fugene. All wereincubated at RT for 15 minutes. 5 ul of each transfection, intriplicate, was added to wells of two, 96-well plates (one+Rapamycin onewithout Rapamycin). 100 μl of HEK293T at 200,000 cells/ml in DMEM+10%FBS were then added to the wells, and the transfected cells incubatedovernight at 37° C.

The medium was then removed from the cells, and the cells washed with200 μl DPBS. 50 μl of 50 nM rapamycin was added, and the cells incubatedfor 1 h at 37° C. 20 μl of 5 mM furimazine in 5 ml phenol red-freeOptiMEMI+50 nM rapamycin was diluted, 50 μl added directly to the cellsand incubated for 5 min in GloMax Multi+. Luminescence was measured onthe GloMax.

FIG. 187 demonstrates that the dark peptides, when fused to FKBP, canreduce the background signal of NLpoly11S (i.e., FRB-NLpoly11S). Takentogether FIGS. 188-190 demonstrate that the dark peptides, when fused toFKBP, can 1) compete with the folding of full length NanoLuc (i.e.,FRB-NanoLuc or NanoLuc-FRB) and 2) compete with both low and highaffinity peptides (also FKBP fusions) for binding to NLpoly11S (i.e.FRB-NLpoly11S), and as a result reduce the total luminescence beingproduced and detected in live cells.

Example 140 Virology Applications

In addition to enabling measurement of viral titers, spontaneouslyinteracting NLpeps also enable studying re-assortment of viruses (e.g.,influenza). Re-assortment of viruses refers to the formation of new“hybrid” viruses from dual infections e.g. H1N1, H5N1, H3N2 (H ishemagglutinin; N is neuraminidase); bird, human, pig, chicken (mostcommon in pigs)

Because of its segmented nature, the influenza genome can be readilyshuffled in host cells infected with more than one virus. When a cell isinfected with influenza viruses from different species, reassortment canresult in progeny viruses that contain genes from strains that normallyinfect birds and genes from strains that normally infect humans, leadingto the creation of new strains that have never been seen in most hosts.Moreover, because at least 16 different subtypes and nine differentneuraminidase subtypes have been characterized, many differentcombinations of capsid proteins are possible. Of these subtypes, threesubtypes of hemagglutinin (H1, H2, and H3) and two subtypes ofneuraminidase (N1 and N2) have caused sustained epidemics in the humanpopulation. Birds are hosts for all influenza A subtypes and are thereservoir from which new HA subtypes are introduced into humans (Palese,2004).

The application of the present system for detecting re-assortment isthat the two components of spontaneously interacting NLpeps are be putinto different viral particles, or the large component in cells and thesmall component in a virus, and the presence of both elements (e.g.,being present in a cell) is detected by luminescence.

Example 141 Validating the Use of Spontaneously Interacting NLpep86 asan Epitope Tag for Proteins Degraded by the Proteasome

Experiments were conducted during development of embodiments of thepresent invention to validate the use of NLpep86 as a tag to monitorexpression levels of proteins degraded by the proteasome. To do this,Nlpep86 was fused to firefly luciferase variants that were also fused toeither one or more PEST, CL1 or ubiquitin sequences (pBC21, 22, 24-29).Each of these constructs is expected to undergo proteasome-mediatedturnover to varying degrees following expression from a mutant CMVpromoter (d1CMV).

The constructs pBC21, 22, 24-29 and control constructs expressinguntagged firefly luciferase or untagged firefly luciferase fused to aPEST sequence (ATG082 and ATG083) were transiently transfected into HELAcells plated at 10,000 cells per well in a 96-well plate using 100 μL ofDMEM+10% FBS. The following day, 10 μL of a transfection mixture (920 μlOptiMEM I+5 ug of the respective construct+15 μl Fugene HD) was addedper well and cells were allowed to incubate for 48 hours in a 37° C.incubator containing 5% CO2. Protein expression levels were quantifiedin replicate wells for each construct by detecting firefly luciferaseactivity or by adding a detection reagent containing NLpoly 11 S(purified NLpoly 11 S added to NanoGlo®). A good correlation wasobserved between the NLpep86 and Fluc signals in each case, suggestingthat NLpep86 detection can be used to monitor fusion protein expressionlevels for proteins degraded by the proteasome.

BC21  (SEQ ID NO: 172) MVSGWRLFKKIS-GGSGGGGSGG-Fluc (SEQ ID NO: 2581) (high affinity) BC22  (SEQ ID NO: 172)MVSGWRLFKKIS-GGSGGGGSGG-FlucP (SEQ ID NO: 2581)  (high affinity) BC24 (SEQ ID NO: 172) pFC15A/MVSGWRLFKKIS-GGSGGGGSGG-Fluc-CL1 (SEQ ID NO: 2581) BC25  (SEQ ID NO: 172)MVSGWRLFKKIS-GGSGGGGSGG-Fluc-PEST12opt (SEQ ID NO: 2581) (high affinity) BC26  (SEQ ID NO: 172)MVSGWRLFKKIS-GGSGGGGSGG-Fluc-CP (SEQ ID NO: 2581) (high affinity) BC27 (SEQ IN NO: 390) UBQ G76V-VGKLGRQDP (SEQ ID NO: 2583)- Fluc (EDAKNIKK . . . (SEQ ID NO: 2582))-GGSGGGGSGG (SEQ ID NO: 2581)-VSGWRLFKKIS (high affinity) BC28  (SEQ IN NO: 390)UBQ-RGKLGRQDP (SEQ ID NO: 2584)-Fluc (EDAKNIKK . . . (SEQ ID NO: 2582))-GGSGGGGSGG (SEQ ID NO: 2581)-VSGWRLFKKIS ((high affinity) BC29 UBQ-LGKLGRQDP (SEQ ID NO: 2585)-Fluc (EDAKNIKK . . . (SEQ ID NO: 2582))-GGSGGGGSGG (SEQ ID NO: 2581)-VSGWRLFKKIS (high affinity)ATG083 D1 FlucP; pF4Ag CMV Luc2-PEST ATG082 D1Fluc; pF4Ag CMV Luc2After a 48 hour incubation, 100 uL NanoGlo® NLpep 11 S reagent (90 μl ofNLpoly11S in 50 ml of NanoGlo® assay reagent was added to each well andincubate for 3 minutes with shaking. Luminescence was then read onGloMax luminometer (0.5 sec/well).

The results in FIG. 191 demonstrate that the signal from Fluc andNLpep86 appear to reflect each other with respect to relative brightnessand have similar RLUs. BC21, BC25 and BC29 are the brightest constructsof the BC series with BC21 appearing the brightest in this experimentBC24, 26 and 27 are the least bright which is predicted from theengineered destabilization.

Example 142

This example demonstrates that a known complementing peptide can be usedas a linker between the same or a different complementing NLpep andNLpoly11S (e.g., NLpep78(2×)).

HEK293T cells (20,000) were transfection with a mixture containing 20 μLof NLpep78-HaloTag (HT) or NLpep78(2×)-HT DNA, 80 μL of phenol-freeOpimex and 8 μL of FugeneHD. Cells were grown overnight at 37° C. andassayed at 24 h using NanoGlo® assay reagent (Promega Corporation)containing 33 nM purified NLpoly 11 S.

The results (FIG. 192) demonstrate that a tandem binding peptide can beused and that it may suffice as a linker.

Example 143 Comparison of the Specific Activities of Wild-TypeOplophorus Luciferase Residues 1-156, NLpoly11S and NanoLuc in HEK293TLysates

Each clone was inserted into pFN21A HaloTag® CMV Flexi® Vector (PromegaG2821), and lysates were prepared as follows: 3 ml HEK293T cells thathave been diluted to a concentration of 200,000 cells/ml (600,000 cellstotal) were plated into each well of a 6-well plate and grown overnightat 37° C. in a CO2 incubator. The following day transfection complexesof each DNA were prepared by combining 6.6 μg of DNA, Opti-MEMO (LifeTechnologies 11058-021) to a final volume of 310 μl and 20 μl of FuGENE®HD (Promega E231a). The transfection complexes were incubated for 20minutes and then 150 μl of each complex added in duplicate to cells. Thecells were grown overnight 37° C. in a CO₂ incubator. The following day,the cells were washed cells with DPBS (Life Technologies 14190-144), and1 ml fresh DPBS added. Cells were frozen to lyse and then thawed fortesting. Duplicate transfection reactions lysates were combined.

To quantitate the level of protein expression for each sample, eachsample was labeled with HaloTag® TMR Ligand (Promega Corporation) asfollows: HaloTag® TMR Ligand (Promega G8251) was diluted 1:100 intowater to a concentration of 0.05 mM; 100 μl of each lysate was mixedwith 2 μl of diluted TMR ligand and incubated for 30 minutes at RT; 20μl of SDS loading dye was added, and the samples heated to 95° C. for 5minutes. 10 μl and 20 μl of each sample was loaded onto a polyacrylamidegel (Bio-Rad, 4-15% Criterion™ Tris-HCl Gel #345-0030.), run at 200V for1 hour and then quantitated using ImageQuant™ LAS 4000 (GE). BothNLpoly11S and NanoLuc® luciferase (Nluc) expressed approximately 4-foldhigher than 1-156.

In order to compare the specific activities of Nluc (full length enzyme)to wt Oplophorus1-156 and NLpoly11S in combination with wt Oplophorus157-169 peptide (binary proteins), substrate titrations were run for allof the samples, but for the binary samples substrate titrations were runat multiple peptide concentrations. Using this format, it was possibleto calculate a Vmax value for Nluc and both Vmax and Bmax values forNLpoly11S and wt Oplophorus 1-156. Three separate experiments were runusing this format and Vmax and Bmax values were normalized to the Vmaxof NanoLuc. Relative specific activities (calculated as averages of Vmaxand Bmax) are normalized to NanoLuc.

Relative specific activity (*with Sample wt 157-169 peptide) NanoLuc1.00 wt 1-156 0.07* 11S 0.18*

Example 144 Effect of NLpoly and NLpep on Intracellular Half-Life ofFlucP

To determine if appending either NLpoly11S or NLpep114 to Luc2-PESTalters the intracellular half-live as measured by decay of signal aftercycloheximide (CHX) treatment.

Day 1: Plate Hela cells in 6 well plates. Plate 3 ml of cells(200,000/ml) into two 6 well plates. Grow overnight. DMEM+10% FBS.

Constructs containing FlucP, wt 157-169 FlucP, NLpoly11S, NLpep114 FlucPand pBC22 (all pF4Ag D1-CMV) were transfected into HeLa cells. Briefly,33 μl of DNA (3.3 ug) was added to 122 μl of OptiMem, mixed and 9.9 μlof FuGENE®HD added. The transfection mixtures were then incubated at RTfor 20 minutes, and 150 μl added to cells. After an overnightincubation, cells were replated at 10,000 cells/well and incubated againovernight.

After incubation, the growth media was removed and replaced with either0.4 mM cycloheximide (CHX) or control (DMSO). At each time point,ONE-Glo™ assay reagent was added, incubated at RT for 3 minutes andluminescence measured on Tecan GENios Pro luminometer.

FIG. 193 demonstrates that none of the NLpoly or NLpep components testedinterfere with the normal intracellular degradation of a reporter enzyme(FlucP).

Example 145 Extracellular Protease Activity Assay

In some embodiments, the present invention provides an assay forextracellular protease (e.g. caspase) activity. A quenched peptide isprovided (e.g., high affinity peptide such as NLpep86) that can only beaccessed and refolded into an active luciferase with an NLpoly, e.g.,NLpoly11S, upon removal of the quencher moiety by a protease (e.g.caspase)(FIG. 194). NLpoly11S and furimazine are introduced to the assayas a reagent and then samples are measured for bioluminescence.

Example 146 Medially Attached Pro-Groups (Isopeptides and GlycosylatedAmino Acids)

Assays are provided for measuring the activity of an enzyme throughusing a ProNLpep. This configuration of ProNLpep is a NLpep with one ofthe internal amino acids conjugated to a group that prevents thecomplementation of the peptide to an NLpoly. When this ProNLpepencounters an enzyme that removes the blocking group (e.g., caspase 1 inthe case of WEHD or a glycosidase in the case of the serine glycoside),the ability of the NLpep to complement to an NLpoly is restored (FIG.195). In the presence of furimazine, this results in production of lightin proportion to the activity of the enzyme of interest. Because eachenzymatic cleavage results in the formation of a luciferase, thesensitivity of this system for assaying small concentrations of enzymeis expected to be high.

Example 147 Linker Evaluation

Assays are provided measuring the release of cargo from an antibody. AnNLpep is attached to an antibody, protein, peptide, or transporterrecognition moiety in such a manner that prevents it from associatingwith a NLpoly to form a luciferase. Upon a stimulus, such as cellularinternalization, the linker between the antibody, protein, peptide, ortransporter recognition moiety and the NLpep is cleaved, due tointracellular reducing potential, and the NLpep is released (FIG. 196).The NLpep can now complement with an NLpoly to form a luciferase, andthe light generated will be proportional to the cleavage of the linker.This provides a system to measure the release of a compound from anantibody, which is a surrogate for cytotoxic drug delivery from AntibodyDrug Conjugates. The linker can be cleaved through any manner known inthe art, such as through intracellular proteases or pH sensitivity.Again, because a luciferase is generated through every cleavage, this isexpected to be a sensitive method for assaying cleavage.

Example 148

The use of antibodies to target and destroy diseased cells has shownsignificant therapeutical promise and occurs through a process calledAntibody-dependent cell-mediated cytotoxicity (ADCC). There are manyways to monitor ADCC activity, including crosslinking of different cellstypes or monitoring gene transcription using specific luciferasereporters expressed in the effector cells. A potential alternativereadout to the ADCC mechanism of action could be in the monitoring ofspecific protein:protein interactions induced or disrupted after thebinding of therapeutical antibodies to their target antigens orreceptors presented on the cell surface. In some embodiments, thespecific protein:protein interactions are monitored using the system ofthe present invention, which provides a readout in the time frame ofminutes versus hours which is required by other methods.

Example 149 Immunoassays

Embodiments of the present invention find use in homogeneousimmunoassays, for example, as depicted in FIG. 201, where the NLpep andNLpoly are fused to binding moieties (e.g., A and B). The bindingmoieties A and B may comprise many different components, making upseveral different formats of immunoassays than can be utilized as targetspecific assays or more generalized reagents to be used in immunoassays.The binding moieties will only come into close proximity in the presenceof the target, thus bringing the NLpep and NLpoly into close proximityresulting in production of luminescence upon substrate addition. Table 7lists exemplary of binding moieties (Mie et al. The Analyst. 2012 Mar.7; 137(5):1085-9.;

Stains et al. ACS chemical biology. 2010 Oct. 15; 5(10):943-52.; Ueda etal. Journal of immunological methods. 2003 August; 279(1-2):209-18. Uedaet al. Nature biotechnology. 1996 December; 14(13):1714-8.; Lim et al.Analytical chemistry. 2007 Aug. 15; 79(16):6193-200.; Komiya et al.Analytical biochemistry. 2004 Apr. 15; 327(2):241-6.; Shirasu et al.Analytical sciences: the international journal of the Japan Society forAnalytical Chemistry. 2009 September; 25(9):1095-100.; hereinincorporated by reference in their entireties).

TABLE 7 Example from (A) binding moiety (B) Binding moiety literatureReference Domain of Protein A Domain of Protein A Rluc fragments fusedMie et al, Analyst, to B-domain of 2012 protein A to detect E. coli withprimary anti-coli Ab + fusion complexed rabbit anti-mouse IgG Protein AProtein A Protein G Protein G Domain of protein G Domain of protein GPolyclonal Ab Polyclonal Ab: either same or second pAB recognizing sametarget mAb mAb to same target recognizing different epitope scFv scFvfrom an antibody Omnitarg and Stains et al, ACS Chem recognizingdifferent Herceptin Fluc fusions Biol, 2010; Komiya et epitope on samefor HER2 detection; b- al, Analytical target gal fusions humanBiochemistry, 2004 serum albumin antibodies Receptor domain 1 Receptordomain 2 to Flt-1 domain 1 and 2 Stains et al, ACS Chem same target Flucfusions for VEGF Biol, 2010 detection Ab variable heavy Ab variablelight chain b-gal chain fusions Ueda et al, J of chain of same antibodyfor HEL; alkaline Immunological phosphatase and Methids, 2003;thioredoxine fusions Shirasu et al, for benzaldehyde AnalyticalSciences, 2009 Mix and match: Mix and match: CD4 receptor domain Stainset al, ACS Chem pAb, mAb, scFv, pAb, mAb, scFv, and scFv of anti- Biol,2010 receptor domain, Vh, receptor domain, Vh, gp120 antibody Fluc VI VIfusions for HIV detection;In some embodiments in which the binding moieties are comprised ofprotein A, protein G, or domains of protein A or G, the immunoassaysystem utilizes the NLpoly and NLpep fusions to complex with antibodiesprior to addition with the sample containing the target. Antibodies bindnon-covalently to protein A and G naturally. Introduction of a covalentcoupling between the antibody and the fusions are introduced in thecomplex formation step. The NLpep/NLpoly-protein A/G/domain fusionsbinding moieties can be complexed to the antibodies in various formats,for example:

-   -   individually with two different specific antibodies targeting        two different proteins to determine if proteins exist in        complex;    -   together with a single target specific polyclonal antibody;    -   together with secondary antibody (e.g., rabbit anti mouse IgG)        to bind to sample preincubated with primary antibody (e.g.,        target specific mouse IgG); and    -   individually with two antibodies targeting two different        epitopes on the same target protein.

As described in Table 7, in some embodiments, the binding moieties aretarget specific antibodies, domains of target specific antibodies,receptor domains that bind target ligands, or a combination ofantibodies, antibody domains, and target receptor domains.

In some embodiments, targets are monitored in samples which include butare not limited to blood, plasma, urine, serum, cell lysates, cells(primary or cell lines), cell culture supernatant, cerebral spinalfluid, bronchial alveolar lavage, tissue biopsy samples, chemicalcompounds, etc.

Methods describe analysis of targets which include but are not limitedto: proteins, small molecules and compounds, haptens, peptides,hormones, heterodimeric protein-protein interactions, cell surfaceantigens, interactions between receptors and ligands, proteins incomplex, viruses and viral components, bacteria, toxins, synthetic andnatural drugs, steroids, catecholamines, eicosanoids, proteinphosphorylation events, etc.

Applications include but are not limited to detection or quantitation oftarget for clinical disease monitoring, diagnostics, therapeutic drugmonitoring, biological research, pharmaceuticals, compound detection andmonitoring in the food/beverage/fragrance industry, viral cladeidentification, etc.

Additional applications include high throughput screening of moleculescapable of disrupting the interactions of target with its receptor thusresulting in a loss of signal assay. There are several proposed formatsfor use of NLpep/NLpoly in immunoassays. In some embodiments, these areperformed homogeneously and supplied as a kit, as separate diagnosticand research kit components, or as stand-alone reagents customizable tothe individual's assay.

In other embodiments, homogeneous immunoassay utilizingNLpep/NLpolyutilize variations of the HitHunter or CEDIA technology(Yang et al. Analytical biochemistry. 2005 January 1; 336(1):102-7.;Golla and Seethala. Journal of biomolecular screening. 2002 December;7(6):515-25.; herein incorporated by reference in their entireties). Insuch assays, components include: target specific antibody, NLpoly,NLpep-recombinant target fusion, and substrate. The NLpoly andNLpep-recombinant target fusion form a luminescent complex when theNLpep is not bound to the target specific antibody. Upon addition of thetest sample to the assay components, the amount of luminescence isdirectly proportional to the target concentration in the test sample asthe target present in the test sample will compete with theNLpep-recombinant target fusion on the antibody (e.g., gain of signalindicates the presence of the target).

Example 150 Exemplary Configurations for NLpoly11S in SpontaneousComplementation

Various configurations of NLpoly11S may find use in spontaneouscomplementation assays or systems. Such configurations may include:deletions at the C-term (e.g., to reduce background luminescence), N-and/or C-terminal appendages (e.g., based on whether they are to bepurified by His or HaloTag), etc. For example, the appendage left byHaloTag when it's a N-terminal tag is SDNIAI. Exemplary configurationsinclude: SDNAIA-11S (HaloTag purification); SDN-11S; SDNAIA-11S, withsingle del at C-term; SDN-11S, with single del at C-term; SDNAIA-11S,with double del at C-term; SDN-11S, with double del at C-term;SDNAIA-11S, with triple del at C-term; SDN-11S, with triple del atC-term; 6His-AIA-11S; 6His-11S; 6His-AIA-11S with single del at C-term;6His-11S with single del at C-term; 6His-AIA-11S with double del atC-term; 6His-11S with double del at C-term; 6His-AIA-11S with triple delat C-term; 6His-11S with triple del at C-term; 11S-6His; 11S-6His, minusC-term 11S residue; 11S-6His, minus last two C-term 11S residues;11S-6His, minus last three C-term 11S residues; 11S-HT7, minus C-term11S residue; 11S-HT7, minus last two C-term 11S residues; 11S-HT7, minuslast three C-term 11S residues; 6His-HT7-AIA-11S; 6His-HT7-11S;6His-AIA-HT7-11S (with single, double, triple 11S C-term dels);6His-HT7-11S (with single, double, triple 11S C-term dels);11S-HT7-6His; 11S-HT7-6His (with single, double, triple 11S C-termdels); and Ternary 11S.

Example 151 Protein Interactions for Binary Complementation Studies

The binary complementation system described herein has been used toanalyze a wide variety of protein interactions (See Table 8).

TABLE 8 Protein Interactions for Binary Complementation StudiesInteraction Status FRB/FKBP Tested V2R/ARRB2 Tested V2R HomodimerizationTested BRD4/H3.3 Tested L3MBTL3/BCLAF1 Tested GR Homodimerization TestedRAS/RAF Tested p53/MDM2 Tested EGFR/GRB2 Tested BCL2/BIM/BAX TestedMYC/MAX Tested CUL1/NEDD8 In Progress EZH2/SUZ12/EED In Progress GABAAMultimerization In Progress

Example 152 Dissociation Constants and Bmax Values for NLpolys with 108Variants of NLpeps (Array#2)

NLpeps were synthesized in array format by New England Peptide (peptidesblocked at N-terminus by acetylation and at C-terminus by amidation;peptides in arrays were synthesized at ˜2 mg scale) (Table 9). Eachpeptide was lyophilized in 2 separate plates. Each well from 1 of theplates of peptides was dissolved in 100 uL nanopure water, and the A260measured and used to calculate the concentration using the extinctioncoefficient of each peptide. The concentration was then adjusted basedon the purity of the peptide, and nanopure water was added to give afinal concentration of 800 uM.

TABLE 9  Peptide array 2 sequences Sequence SEQ ID NO. array2.1VTGYRLFKKIS 2462 array2.2 VTGYRLFKKAS 2463 array2.3 VTGYRLFKKES 2464array2.4 VTGYRLFKQIS 2465 array2.5 VTGYRLFKQAS 2466 array2.6 VTGYRLFKQES2467 array2.7 VTGYRLFKEIS 2468 array2.8 VTGYRLFKEAS 2469 array2.9VTGYRLFKEES 2470 array2.10 VTGYRLFQKIS 2471 array2.11 VTGYRLFQKAS 2472array2.12 VTGYRLFQKES 2473 array2.13 VTGYRLFQQIS 2474 array2.14VTGYRLFQQAS 2475 array2.15 VTGYRLFQQES 2476 array2.16 VTGYRLFQEIS 2477array2.17 VTGYRLFQEAS 2478 array2.18 VTGYRLFQEES 2479 array2.19VTGYRLFEKIS 2480 array2.20 VTGYRLFEKAS 2481 array2.21 VTGYRLFEKES 2482array2.22 VTGYRLFEQIS 2483 array2.23 VTGYRLFEQAS 2484 array2.24VTGYRLFEQES 2485 array2.25 VTGYRLFEEIS 2486 array2.26 VTGYRLFEEAS 2487array2.27 VTGYRLFEEES 2488 array2.28 VTGYRLFKKIL 2489 array2.29VTGYRLFKKAL 2490 array2.30 VTGYRLFKKEL 2491 array2.31 VTGYRLFKQIL 2492array2.32 VTGYRLFKQAL 2493 array2.33 VTGYRLFKQEL 2494 array2.34VTGYRLFKEIL 2495 array2.35 VTGYRLFKEAL 2496 array2.36 VTGYRLFKEEL 2497array2.37 VTGYRLFQKIL 2498 array2.38 VTGYRLFQKAL 2499 array2.39VTGYRLFQKEL 2500 array2.40 VTGYRLFQQIL 2501 array2.41 VTGYRLFQQAL 2502array2.42 VTGYRLFQQEL 2503 array2.43 VTGYRLFQEIL 2504 array2.44VTGYRLFQEAL 2505 array2.45 VTGYRLFQEEL 2506 array2.46 VTGYRLFEKIL 2507array2.47 VTGYRLFEKAL 2508 array2.48 VTGYRLFEKEL 2509 array2.49VTGYRLFEQIL 2510 array2.50 VTGYRLFEQAL 2511 array2.51 VTGYRLFEQEL 2512array2.52 VTGYRLFEEIL 2513 array2.53 VTGYRLFEEAL 2514 array2.54VTGYRLFEEEL 2515 array2.55 VEGYRLFKKIS 2516 array2.56 VEGYRLFKKAS 2517array2.57 VEGYRLFKKES 2518 array2.58 VEGYRLFKQIS 2519 array2.59VEGYRLFKQAS 2520 array2.60 VEGYRLFKQES 2521 array2.61 VEGYRLFKEIS 2522array2.62 VEGYRLFKEAS 2523 array2.63 VEGYRLFKEES 2524 array2.64VEGYRLFQKIS 2525 array2.65 VEGYRLFQKAS 2526 array2.66 VEGYRLFQKES 2527array2.67 VEGYRLFQQIS 2528 array2.68 VEGYRLFQQAS 2529 array2.69VEGYRLFQQES 2530 array2.70 VEGYRLFQEIS 2531 array2.71 VEGYRLFQEAS 2532array2.72 VEGYRLFQEES 2533 array2.73 VEGYRLFEKIS 2534 array2.74VEGYRLFEKAS 2535 array2.75 VEGYRLFEKES 2536 array2.76 VEGYRLFEQIS 2537array2.77 VEGYRLFEQAS 2538 array2.78 VEGYRLFEQES 2539 array2.79VEGYRLFEEIS 2540 array2.80 VEGYRLFEEAS 2541 array2.81 VEGYRLFEEES 2542array2.82 VEGYRLFKKIL 2543 array2.83 VEGYRLFKKAL 2544 array2.84VEGYRLFKKEL 2545 array2.85 VEGYRLFKQIL 2546 array2.86 VEGYRLFKQAL 2547array2.87 VEGYRLFKQEL 2548 array2.88 VEGYRLFKEIL 2549 array2.89VEGYRLFKEAL 2550 array2.90 VEGYRLFKEEL 2551 array2.91 VEGYRLFQKIL 2552array2.92 VEGYRLFQKAL 2553 array2.93 VEGYRLFQKEL 2554 array2.94VEGYRLFQQIL 2555 array2.95 VEGYRLFQQAL 2556 array2.96 VEGYRLFQQEL 2557array2.97 VEGYRLFQEIL 2558 array2.98 VEGYRLFQEAL 2559 array2.99VEGYRLFQEEL 2560 array2.100 VEGYRLFEKIL 2561 array2.101 VEGYRLFEKAL 2562array2.102 VEGYRLFEKEL 2563 array2.103 VEGYRLFEQIL 2564 array2.104VEGYRLFEQAL 2565 array2.105 VEGYRLFEQEL 2566 array2.106 VEGYRLFEEIL 2567array2.107 VEGYRLFEEAL 2568 array2.108 VEGYRLFEEEL 2569

Peptides were diluted to 400 uM (4×) in PBS+0.1% Prionex and thendiluted serially 7 times (8 concentrations total) in 0.5 log steps(3.162 fold dilution). NLpoly 11S was diluted 1:10-6 into PBS+0.1%Prionex. 25 uL each NLpep+25 uL NLpoly 11S were mixed and incubated for30 min at RT. 50 uL NanoGlo+100 uM Fz was added and incubated for 30 minat RT. Luminescence was measured on a GloMax Multi+ with 0.5 secintegration. Kd/Bmax were determined using Graphpad Prism, Onesite-specific binding, best-fit values. Table 10 indicates thedissociation constant and Bmax values for NLpoly 11S and the indicatedNLPep. The results indicate the affects of mutations on the binding toNLpoly 11S and the ability of the complex to produce luminescence.

TABLE 10 SEQ ID Peptide NO: Sequence Bmax Kd Bmax Kd array2.1 2462VTGYRLFKKIS 134567 0.01334 4936 0.003695 array2.2 2463 VTGYRLFKKAS103904 0.2411 711.8 0.006084 array2.3 2464 VTGYRLFKKES 55963 0.773 17050.06499 array2.4 2465 VTGYRLFKQIS 104275 0.7462 4670 0.09318 array2.52466 VTGYRLFKQAS 31031 1.953 436.4 0.05649 array2.6 2467 VTGYRLFKQES5006 1.348 182 0.1583 array2.7 2468 VTGYRLFKEIS 32026 4.438 1173 0.5196array2.8 2469 VTGYRLFKEAS 3929 2.568 200.6 0.3566 array2.9 2470VTGYRLFKEES 1453 3.863 118.6 1.044 array2.10 2471 VTGYRLFQKIS 1125400.08118 4037 0.01352 array2.11 2472 VTGYRLFQKAS 80943 0.7485 4035 0.1039array2.12 2473 VTGYRLFQKES 17237 0.4173 3190 0.3233 array2.13 2474VTGYRLFQQIS 19401 0.876 2357 0.47 array2.14 2475 VTGYRLFQQAS 4351 1.111311.1 0.3392 array2.15 2476 VTGYRLFQQES 5197 7.486 198.7 0.797 array2.162477 VTGYRLFQEIS 1321 2.561 112.6 0.5939 array2.17 2478 VTGYRLFQEAS NDND ND ND array2.18 2479 VTGYRLFQEES 5112 67.32 426.5 11.22 array2.192480 VTGYRLFEKIS 122961 0.6047 11827 0.2689 array2.20 2481 VTGYRLFEKAS36284 1.794 935.8 0.09793 array2.21 2482 VTGYRLFEKES 8622 1.491 599.70.3267 array2.22 2483 VTGYRLFEQIS 121402 10.78 3711 1.121 array2.23 2484VTGYRLFEQAS 3824 4.174 243.4 0.8621 array2.24 2485 VTGYRLFEQES 18297.832 24.45 0.2891 array2.25 2486 VTGYRLFEEIS ND ND ND ND array2.26 2487VTGYRLFEEAS ND ND ND ND array2.27 2488 VTGYRLFEEES ND ND ND ND array2.282489 VTGYRLFKKIL 140640 0.07664 6033 0.02 array2.29 2490 VTGYRLFKKAL98575 0.2755 1679 0.0168 array2.30 2491 VTGYRLFKKEL 51143 0.6714 20000.07542 array2.31 2492 VTGYRLFKQIL 115248 2.989 2995 0.3361 array2.322493 VTGYRLFKQAL 34875 3.561 496 0.1247 array2.33 2494 VTGYRLFKQEL 85481.953 581.1 0.5209 array2.34 2495 VTGYRLFKEIL 21933 4.405 867.2 0.7072array2.35 2496 VTGYRLFKEAL 5547 5.153 180.1 0.6609 array2.36 2497VTGYRLFKEEL 1720 7.785 75.68 1.256 array2.37 2498 VTGYRLFQKIL 1274040.3625 7870 0.1108 array2.38 2499 VTGYRLFQKAL 72788 0.9748 3853 0.1796array2.39 2500 VTGYRLFQKEL 33109 2.477 687.6 0.1414 array2.40 2501VTGYRLFQQIL 66256 122.3 13366 40.42 array2.41 2502 VTGYRLFQQAL 3472 3.97314 1.484 array2.42 2503 VTGYRLFQQEL 14230 18.99 180.8 0.714 array2.432504 VTGYRLFQEIL 9406 17.25 544.2 2.141 array2.44 2505 VTGYRLFQEAL 423315.99 426.5 4.994 array2.45 2506 VTGYRLFQEEL 14254 35.43 614.2 3.766array2.46 2507 VTGYRLFEKIL 219381 1.917 7349 0.2965 array2.47 2508VTGYRLFEKAL 34526 1.807 1377 0.216 array2.48 2509 VTGYRLFEKEL 108652.437 823.9 0.5103 array2.49 2510 VTGYRLFEQIL 99205 124.3 2780 5.68array2.50 2511 VTGYRLFEQAL 17117 40.4 294 1.642 array2.51 2512VTGYRLFEQEL 46162 85 1436 4.881 array2.52 2513 VTGYRLFEEIL 15703 104.1560 6.409 array2.53 2514 VTGYRLFEEAL ND ND ND ND array2.54 2515VTGYRLFEEEL 251166 68.27 15593 5.901 array2.55 2516 VEGYRLFKKIS 423840.07805 3011 0.02593 array2.56 2517 VEGYRLFKKAS 15920 0.6409 510.20.05975 array2.57 2518 VEGYRLFKKES 3374 0.891 142.5 0.1335 array2.582519 VEGYRLFKQIS 21512 2.091 665.9 0.244 array2.59 2520 VEGYRLFKQAS 23002.088 74.01 0.1938 array2.60 2521 VEGYRLFKQES 4346 10.64 91.51 0.7646array2.61 2522 VEGYRLFKEIS 5459 14.43 116.8 0.7024 array2.62 2523VEGYRLFKEAS 2375 22.05 112.3 2.964 array2.63 2524 VEGYRLFKEES 17264220.3 3074 54.34 array2.64 2525 VEGYRLFQKIS 36517 0.5863 781.4 0.05853array2.65 2526 VEGYRLFQKAS 10620 1.929 271.7 0.1454 array2.66 2527VEGYRLFQKES 3489 2.87 132.3 0.3846 array2.67 2528 VEGYRLFQQIS 5223 8.143199.6 0.8457 array2.68 2529 VEGYRLFQQAS 3753 20.01 117.8 1.833 array2.692530 VEGYRLFQQES ND ND ND ND array2.70 2531 VEGYRLFQEIS 29161 230.2560.4 6.062 array2.71 2532 VEGYRLFQEAS 44893 24.03 1778 1.825 array2.722533 VEGYRLFQEES ND ND ND ND array2.73 2534 VEGYRLFEKIS 22544 2.148641.3 0.2291 array2.74 2535 VEGYRLFEKAS 3808 4.138 122.2 0.3119array2.75 2536 VEGYRLFEKES 1170 2.969 136.7 1.282 array2.76 2537VEGYRLFEQIS 17957 52.79 724 4.614 array2.77 2538 VEGYRLFEQAS 26862 48.29436.5 1.752 array2.78 2539 VEGYRLFEQES 39375 252.3 4842 41.61 array2.792540 VEGYRLFEEIS ND ND ND ND array2.80 2541 VEGYRLFEEAS 383183 1419572696 2258 array2.81 2542 VEGYRLFEEES ND ND ND ND array2.82 2543VEGYRLFKKIL 43371 0.563 2640 0.16 array2.83 2544 VEGYRLFKKAL 20849 1.588591.2 0.1396 array2.84 2545 VEGYRLFKKEL 7828 1.413 ND ND array2.85 2546VEGYRLFKQIL 31425 10.34 358.7 0.2986 array2.86 2547 VEGYRLFKQAL 23042.274 54.26 0.2428 array2.87 2548 VEGYRLFKQEL 1790 12.59 113 2.614array2.88 2549 VEGYRLFKEIL 9831 17.75 551.8 3.002 array2.89 2550VEGYRLFKEAL 5574 42.42 435.1 7.715 array2.90 2551 VEGYRLFKEEL 12241100.9 458.5 6.589 array2.91 2552 VEGYRLFQKIL 50503 2.077 2173 0.3373array2.92 2553 VEGYRLFQKAL 12294 2.023 430.5 0.206 array2.93 2554VEGYRLFQKEL 4090 1.691 278.5 0.4617 array2.94 2555 VEGYRLFQQIL 2281 9.39112 1.201 array2.95 2556 VEGYRLFQQAL 38229 18.81 1578 1.617 array2.962557 VEGYRLFQQEL 104621 99.4 6265 10.43 array2.97 2558 VEGYRLFQEIL ND NDND ND array2.98 2559 VEGYRLFQEAL 2696 99.9 238.8 15.53 array2.99 2560VEGYRLFQEEL ND ND ND ND array2.100 2561 VEGYRLFEKIL 34989 10.56 17471.801 array2.101 2562 VEGYRLFEKAL 6372 12.62 186 0.8756 array2.102 2563VEGYRLFEKEL 961.5 5.786 67.06 1.216 array2.103 2564 VEGYRLFEQIL ND ND NDND array2.104 2565 VEGYRLFEQAL 9882 335.8 544.7 23.35 array2.105 2566VEGYRLFEQEL ND ND ND ND array2.106 2567 VEGYRLFEEIL ND ND ND NDarray2.107 2568 VEGYRLFEEAL ND ND ND ND array2.108 2569 VEGYRLFEEEL NDND ND ND

Example 153 Dark Peptides and Quencher Peptides for Reducing BackgroundSignal from NLpoly11S

A purified sample of NLpoly11S was diluted into NanoGlo reagent to givea final concentration of 2 uM. Pep86 is a high affinity luminogenicpeptide and was used to induce maximum signal for NLpoly11S. Pep86 wasprepared at 1 nM in PBS (pH 7.2) for a working solution. Dark peptideand quencher peptides (FIG. 180) were dissolved to 1 mM (or lower) ineither PBS pH 7.2 or 150 mM NH4HCO3 and added in equal volume to theNanoGlo/NLpoly11S and then samples were read on a Tecan Infinite F500reader using a 5 min time point.

FIG. 202A shows that both GWALFKK (SEQ ID NO: 2351) and Dabcyl-GWALFKK(SEQ ID NO: 2351) reduce the background luminescence generated byNLpoly11S in the absence of any other luminogenic peptide. FIG. 202Bshows that Pep86 is able to induce luminescence even in the presence ofGWALFKK (SEQ ID NO: 2351) and Dabcyl-GWALFKK (SEQ ID NO: 2351).

FIG. 203A shows that VTGWALFEEIL (SEQ ID NO: 2372) (Trp 11mer) andVTGYALFEEIL (SEQ ID NO: 2355) (Tyr 11mer) induce luminescence overbackground (NLpoly11S alone; no peptide control), but that theN-terminal Dabcyl versions of each provide significant quenching of thissignal. FIG. 203B shows that Pep86 is able to induce luminescence evenin the presence of the Dabcyl versions of Trp 11mer and Tyr 11mer.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific embodiments, it should be understood that the invention asclaimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the described modes for carrying outthe invention that are obvious to those skilled in the relevant fieldsare intended to be within the scope of the present invention.

1. A peptide comprising an amino acid sequence having less than 100% andgreater than 40% sequence identity with SEQ ID NO: 2, wherein adetectable bioluminescent signal is produced in the presence of asubstrate when the peptide contacts a polypeptide consisting of SEQ IDNO:
 440. 2. The peptide of claim 1, wherein the detectablebioluminescent signal is produced in the presence of a substrate whenthe peptide associates with the polypeptide consisting of SEQ ID NO:440.
 3. The peptide of claim 1, wherein the peptide exhibits enhancementof one or more traits compared to a peptide of SEQ ID NO: 2, wherein thetraits are selected from: affinity for the polypeptide consisting of SEQID NO: 440, expression, intracellular solubility, intracellularstability, and bioluminescent activity when combined with thepolypeptide consisting of SEQ ID NO:
 440. 4. The peptide of claim 1,wherein the amino acid sequence is selected from the peptides ofTable
 1. 5. The peptide of claim 1, wherein the amino acid sequence issynthetic, contains non-natural amino acids or is a peptide mimic.
 6. Anucleic acid comprising a sequence coding for a peptide of claim
 1. 7. Afusion polypeptide comprising the peptide of claim 1 and a firstinteraction polypeptide that is configured to form a complex with asecond interaction polypeptide upon contact of the first interactionpolypeptide and the second interaction polypeptide.
 8. A nucleic acidcomprising a sequence coding for a fusion polypeptide of claim
 7. 9. Abioluminescent complex comprising: (a) the fusion polypeptide of claim7; and (b) a second fusion polypeptide comprising: (i) the secondinteraction polypeptide, and (ii) a complement polypeptide that emits adetectable bioluminescent signal in the presence of a substrate whenassociated with the peptide comprising an amino acid sequence havingless than 100% and greater than 40% sequence identity with SEQ ID NO: 2;wherein the first fusion polypeptide and second fusion polypeptide areassociated; and wherein the peptide comprising an amino acid sequencehaving less than 100% and greater than 40% sequence identity with SEQ IDNO: 440 and the complement polypeptide are associated.
 10. A polypeptidecomprising an amino acid sequence having less than 100% and greater than40% sequence identity with SEQ ID NO: 440, wherein a detectablebioluminescent signal is produced in the presence of a substrate whenthe polypeptide contacts a peptide consisting of SEQ ID NO:
 2. 11. Thepolypeptide of claim 9, wherein the polypeptide exhibits enhancement ofone or more traits compared to a polypeptide of SEQ ID NO: 440, whereinthe traits are selected from: affinity for the peptide consisting of SEQID NO: 2, expression, intracellular solubility, intracellular stability,and bioluminescent activity when combined with the peptide consisting ofSEQ ID NO:
 2. 12. The polypeptide of claim 10, wherein the amino acidsequence is selected from one of the polypeptide sequences of Table 2.13. The polypeptide of claim 10, wherein the detectable bioluminescentsignal is produced in the presence of a substrate when the polypeptideassociates with the peptide consisting of SEQ ID NO:
 2. 14. Thepolypeptide of claim 10, wherein the amino acid sequence is synthetic,contains non-natural amino acids or is a peptide mimic.
 15. A nucleicacid comprising a sequence coding for a polypeptide of claim
 10. 16. Afusion polypeptide comprising the polypeptide of claim 10 and a firstinteraction polypeptide that is configured to form a complex with asecond interaction polypeptide upon contact of the first interactionpolypeptide and the second interaction polypeptide.
 17. A bioluminescentcomplex comprising: (a) the fusion polypeptide of claim 16; and (b) asecond fusion polypeptide comprising: i) the second interactionpolypeptide, and ii) a complement peptide that causes the polypeptidecomprising an amino acid sequence having less than 100% and greater than40% sequence identity with SEQ ID NO: 2 to emit a detectablebioluminescent signal in the presence of a substrate when an associationis formed between the two; wherein the first fusion polypeptide andsecond fusion polypeptide are associated; and wherein the polypeptidecomprising an amino acid sequence having less than 100% and greater than40% sequence identity with SEQ ID NO: 440 and the complement peptide areassociated.
 18. A bioluminescent complex comprising: (a) a peptidecomprising a peptide amino acid sequence having less than 100% andgreater than 40% sequence identity with SEQ ID NO: 2; and (b) apolypeptide comprising a polypeptide amino acid sequence having lessthan 100% and greater than 40% sequence identity with SEQ ID NO: 440,wherein the bioluminescent complex exhibits detectable luminescence. 19.The bioluminescent complex of claim 18, wherein the peptide amino acidsequence and the polypeptide amino acid sequence are associated.
 20. Thebioluminescent complex of claim 18, wherein the peptide amino acidsequence is selected from the peptide sequences of Table
 1. 21. Thebioluminescent complex of claim 18, wherein the polypeptide amino acidsequence is selected from the polypeptide sequences of Table
 2. 22. Thebioluminescent complex of claim 19, wherein the peptide amino acidsequence is attached to a first interaction element that is associatedwith a second interaction element.
 23. The bioluminescent complex ofclaim 22, wherein the polypeptide amino acid sequence is attached to thesecond interaction element.
 24. The bioluminescent complex of claim 23,wherein the polypeptide amino acid sequence and peptide amino acidsequence are incapable of associating in the absence of the associationof the first and second interaction elements.
 25. A bioluminescentcomplex comprising: (a) a first amino acid sequence that is not afragment of a preexisting protein; and (b) a second amino acid sequencethat is not a fragment of a preexisting protein, wherein thebioluminescent complex exhibits detectable luminescence, wherein thefirst amino acid sequence and the second amino acid sequence areassociated, and wherein the bioluminescent complex emits a detectablebioluminescent signal in the presence of a substrate when the firstamino acid sequence and the second amino acid sequence are associated.26. The bioluminescent complex of claim 25, further comprising: (c) athird amino acid sequence comprising a first member of an interactionpair, wherein the third amino acid sequence is covalently attached tothe first amino acid sequence; and (d) a fourth amino acid sequencecomprising a second member of an interaction pair, wherein the fourthamino acid sequence is covalently attached to the second amino acidsequence.
 27. The bioluminescent complex of claim 26, wherein thenon-covalent interactions between the first amino acid sequence and thesecond amino acid sequence are not sufficient to associate the firstamino acid sequence and the second amino acid sequence in the absence ofthe non-covalent interactions between the first member and the secondmember of the interaction pair.
 28. The bioluminescent complex of claim26, wherein a first polypeptide chain comprises the first amino acidsequence and the third amino acid sequence, and wherein a secondpolypeptide chain comprises the second amino acid sequence and thefourth amino acid sequence.
 29. The bioluminescent complex of claim 28,wherein the first polypeptide chain and the second polypeptide chain areexpressed within a cell.
 30. A bioluminescent complex comprising: (a) anon-luminescent pair, wherein each non-luminescent element of thenon-luminescent pair is not a fragment of a preexisting protein; (b) aninteraction pair, wherein each interaction element of the interactionpair is covalently attached to one of the non-luminescent elements ofthe non-luminescent pair.
 31. A method of detecting a stable interactionbetween a first amino acid sequence and a second amino acid sequencecomprising: (a) attaching the first amino acid sequence to a third aminoacid sequence, and attaching the second amino acid sequence to a fourthamino acid sequence, wherein the third and fourth amino acid sequencesare not fragments of a preexisting protein, wherein a stable complex ofthe third and fourth amino acid sequences emits a detectablebioluminescent signal in the presence of a substrate, wherein thenon-covalent interactions between the third and fourth amino acidsequences are insufficient to form a complex of the third and fourthamino acid sequences in the absence of additional stabilizing and/oraggregating forces, and wherein a stable interaction between the firstamino acid sequence and the second amino acid sequence provides theadditional stabilizing and/or aggregating forces to produce a stablecomplex of the third and fourth amino acid sequences; (b) placing thefirst, second, third, and fourth amino acid sequences of step (a) inconditions to allow for stable interactions between the first amino acidsequence and the second amino acid sequence to occur; and (c) detectingthe bioluminescent signal emitted, in the presence of a substrate, bythe stable complex of the third and fourth amino acid sequences in thepresence of a substrate, wherein detection of the bioluminescent signalindicates a stable interaction between the first amino acid sequence andthe second amino acid sequence.
 32. The method of claim 31, whereinattaching the first amino acid sequence to the third amino acid sequenceand the second amino acid sequence to the fourth amino acid sequencecomprises forming a first fusion protein comprising the first amino acidsequence and the third amino acid sequence and forming a second fusionprotein comprising the second amino acid sequence and the fourth aminoacid sequence.
 33. The method of claim 32, wherein the first fusionprotein and the second fusion protein further comprise linkers betweensaid first and third amino acid sequences and said second and fourthamino acid sequences, respectively.
 34. The method of claim 32, whereinthe first fusion protein is expressed from a first nucleic acid sequencecoding for the first and third amino acid sequences, and the secondfusion protein is expressed from a second nucleic acid sequence codingfor the second and fourth amino acid sequences.
 35. The method of claim34, wherein a single vector comprises the first nucleic acid sequenceand the second nucleic acid sequence.
 36. The method of claim 34,wherein the first nucleic acid sequence and the second nucleic acidsequence are on separate vectors.
 37. The method of claim 34, whereinsteps (a) and (b) comprise expressing the first and second fusionproteins within a cell.
 38. A method of optimizing a non-luminescentpair comprising: (a) aligning the sequences of three or more relatedproteins; (b) determining a consensus sequence for the related proteins;(c) providing first and second fragments of a protein related to threeor more proteins, wherein the fragments are individually substantiallynon-luminescent but exhibit luminescence upon stable interaction of thefragments; (d) mutating the first and second fragments at one or morepositions each, wherein said mutations alter the sequences of thefragments to be more similar to a corresponding portion of the consensussequence, wherein the mutating results in a non-luminescent pair thatare not fragments of a preexisting protein, (e) testing thenon-luminescent pair for the absence of luminescence when unassociatedand luminescence upon stable association of the non-luminescent pair.39. The method of claim 38, wherein the non-luminescent pair exhibitsenhancement of one or more traits compared to the first and secondfragments, wherein the traits are selected from: increasedreconstitution affinity, decreased reconstitution affinity, enhancedexpression, increased intracellular solubility, increased intracellularstability, and increased intensity of reconstituted luminescence.
 40. Adetection reagent comprising: (a) a polypeptide comprising an amino acidsequence having less than 100% and greater than 40% sequence identitywith SEQ ID NO: 440, wherein a detectable bioluminescent signal isproduced, in the presence of a substrate, when the polypeptide contactsa peptide consisting of SEQ ID NO: 2, and (b) a substrate for abioluminescent complex produced by said polypeptide and a peptideconsisting of SEQ ID NO:
 2. 41. A detection reagent comprising: (a) apeptide comprising an amino acid sequence having less than 100% andgreater than 40% sequence identity with SEQ ID NO: 2, wherein adetectable bioluminescent signal is produced, in the presence of asubstrate, when the peptide contacts a polypeptide consisting of SEQ IDNO: 440, and (b) a substrate for a bioluminescent complex produced bysaid peptide and a polypeptide consisting of SEQ ID NO: 440.